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.