Discussion
Aging is usually accompanied by low levels of energy and frailty. In all
the aerobic organisms’ mitochondria are the main source of energy
production. For generation of energy, they rely on the availability of
oxygen. However, with age, due to various reasons, including reduced
lung capacity, hematocrit, cardiac output, and capillary density the
oxygen supply to the brain and other organs of the body gets
considerably reduced (Ances et al., 2009; Moeini et al., 2018). Low
oxygen levels further contribute significantly to mitochondrial
dysfunction and aging. Although all the organs get stressed due to low
oxygen availability, but brain is most prone to its adverse effects.
Some PET scan-based studies have conclusively shown that the cerebral
metabolic rate of oxygen (CMRO2) is significantly reduced in the aged
brain tissue due to reduced oxygen availability (Goyal et al., 2017).
Therefore, in this study, we asked whether we can delay the process of
brain aging by pharmacologically increasing the availability of oxygen
to the brain. Though, we also analyzed its effect on other vital orans
of the body as well.
We used crocetin, a known oxygen diffusion enhancer to treat aged mice
and analyze its effect on delaying brain aging. The mice treated for
four months with crocetin did not show any stress and toxicity, which
was evident by no adverse changes in the behavior, body weight, blood
biochemistry and hematological parameters. The analysis of cognitive
behavior indicated a highly significant shift in the crocetin group from
the normally aged untreated mice. Crocetin-treated mice showed
significant improvement in both working and spatial memory behavior,
apart from performing well in the exploratory behavior.
We further wanted to explore the molecular changes behind the
improvement in memory and exploratory behavior. Hippocampus is one of
the most important regions of the brain responsible for processing and
storage of long-term memory, though its involvement in short-term memory
is also being revealed (Hannula, Tranel & Cohen, 2006). Therefore, we
performed the whole transcriptome analysis of hippocampi to identify the
differential change in the gene expression after crocetin treatment.
Transcriptome analysis clearly revealed that multiple genes were
differentially expressed in the hippocampi of the crocetin group, which
was correlated with behavior data and other parameters. These data
further emphasized to explore the exact genes that may be responsible
for changes in memory behavior. We found that some of the most
significantly affected genes in crocetin-treated mice belong to the
mitochondrial genome. Out of the 13 protein-coding genes of the
mitochondrial genome, nine genes were significantly upregulated, which
included ND1, ND2, ND4, ND4L, ND5, ND6, CYTB, ATP6, and ATP8. These
genes play an important role in the ETC and hence contribute to the
production of energy and affect the energy state of the cell.
Interestingly, during aging, the subunits of OXPHOS encoded by the
mitochondrial genome are specifically downregulated rather than the
subunits encoded by the nuclear genome (Gomes et al., 2013). However, we
found that the nuclear gene ubiquinol cytochrome c reductase (Uqcrq),
which is a part of complex III of ETC, was also significantly
upregulated in crocetin-treated mice. Moreover, we found that most of
the pathways affected by crocetin belong to energy metabolism indicating
its strong effect on OXPHOS. Interestingly, OXPHOS is the main target of
most of the life span extension interventions (Tyshkovskiy et al.,
2019). Crocetin also appeared to reverse brain aging by upregulating the
OXPHOS. The highly significant increase in NAD+ and
ATP levels in the brain of crocetin-treated mice also supported these
data. We also observed a significant rise in glucose levels in the
brain, indicating that increased availability of oxygen by crocetin
helped mitochondrial to produce energy more efficiently from glucose and
helped restoring the glucose stores in the brain. This observation is
important as mitochondrial dysfunction during neurodegeneration is
linked to less efficient glucose metabolism by glycolysis (Yao, Irwin,
Zhao, Nilsen, Hamilton & Brinton, 2009).
We hypothesized that the ability of crocetin to enhance the diffusion of
oxygen to the tissues might be contributing to the increased expression
of ETC genes and the generation of more NAD+ and ATP.
Due to the high demand of energy, neurons have a large number of
mitochondria, which require continuous supply of oxygen to produce ATP
(Oruganty-Das, Ng, Udagawa, Goh & Richter, 2012). Our data indicated
that treatment with crocetin helped the old brain cells to meet the
demand of oxygen and regain the mitochondrial activity that could
sustain different cellular functions. The declining level of
NAD+ have been reported to create hypoxia-like
conditions in the cells, which show altered metabolic activities.
Further, these conditions may not be reversible even if the oxygen is
present in abundance (Gomes et al., 2013). However, we found that
increased availability of oxygen by chronic treatment with crocetin
restored the mitochondrial activity and production of energy metabolites
NAD+ and ATP in the brain cells. Further, few SNPs and
InDels in mitochondrial have been linked with mitochondrial dysfunction
and development of aging (Park & Larsson, 2011). However, we did not
find any change in the pattern of SNPs and InDels, which further
supported the increased availability of oxygen as a primary cause for
improved mitochondrial function in the animals treated with crocetin.
Further, we compared the hippocampi gene expression profile of young
mice with old and crocetin-treated mice. In cluster diagram, we found
that the gene expression profile of young mice is closer to
crocetin-treated mice. Additionally, the expression of mitochondrial
genes was significantly high in young mice in comparison to old mice,
however, crocetin treatment of old mice narrowed down this difference.
These findings were also confirmed by the western blotting of two of the
key protein ND5 and ND6, from NADH dehydrogenase complex I of ETC. These
data further emphasized the anti-brain aging effect of crocetin.
Additionally, a few important genes such as Bdnf, Gabbr2, Gad2, and
Drosha that play a vital role in neuronal survival, growth, plasticity
and neurotransmission (Podyma, Parekh, Guler & Deppmann, 2021), often
show declining expression with aging (Kronenberg et al., 2021). However,
we found that the expression of these genes was significantly
upregulated in crocetin-treated mice, which was significantly closer to
young than old mice.
The transcriptome data and the analysis of selective proteins clearly
implicated mitochondria as the main target of crocetin. The reduction in
the mitochondrial potential is a clear indicator of reduced
mitochondrial activity and, thus production of energy, which is one of
the hallmarks of aging (Leuner et al., 2007). Therefore, we intended to
capture the effect of crocetin on the activity of mitochondria in aged
primary astrocytes in the live cell assay for mitochondrial membrane
potential. Being abundantly present in the brain, astrocytes contribute
significantly to preserve physiological homeostasis in the brain (Khakh
& Sofroniew, 2015; Verkhratsky & Nedergaard, 2018). We found that
crocetin exerted its effect on a specific population of cells, among
others, which had relatively low mitochondrial potential. The raised
mitochondrial potential of these cells in the presence of crocetin also
reflected in increased energy levels of the aged astrocytes. These data
again emphasized the anti-brain aging effect of crocetin that comes
through the improved function of mitochondria in aged cells or animals.
The anti-aging effect of crocetin was not confined to brain only, the
analysis of energy metabolites in other vital organs viz. lungs, liver,
heart and kidney of the mice treated with crocetin also showed
significantly higher levels of ATP and NAD+. These
data clearly indicated that increased availability of oxygen affected
the energy production pathways similarly across all tissues of the body.
These data were further supported by improved neuromuscular
co-ordination and muscular strength in mice treated with crocetin.
Moreover, crocetin mediated improvement in energy metabolism through
increased availability of oxygen also enhanced the median life span of
mice. Our findings support the Tromsø study, which indicates that normal
oxygen saturation levels are strongly linked to longevity (Vold, Aasebo,
Wilsgaard & Melbye, 2015).
In conclusion, this study finds crocetin to be a potential lead molecule
against brain and body aging. Crocetin prevents aging and increased
median life span possibly by a unique mechanism of enhancing oxygen
diffusion to the tissues. The increased oxygen helped rescue
mitochondrial dysfunction, improve ETC activity and production of
energy. Our data suggest that similar to calorie restriction, energy
restoration can also have an anti-aging and longevity-enhancing effects.
We believe that crocetin is worth exploring further for the possible
development as a new drug against the neurodegenerative and other
age-related diseases.