Brain Proteome And Antioxidant Regulation Of Hibernating Bats

Bats (order Chiroptera) are the only mammalian group capable of powered flight. Based on traditional taxonomy, bats are divided into suborder Megachiroptera and Microchiroptera. Megabats eat fruits and nectar and they cannot hibernate, while microbats are mainly lived on insects and most of them can hibernate. However, molecular phylo genetic studies redivide bats into suborder Yinpterochiroptera and Yangochiroptera, and both suborders contain hibernating bats. In order to overcome food limitation and high energy demands during winter periods, some animals have evolved hibernation strategy. During hibernation, animals experienced several physiological fluctuations, such as body temperature, metabolic rate, heart rate, and blood flow rate. Since hibernators can tolerate these fluctuations which are lethal to non-hibernators, they become the research interests of many researchers. However, the mechanisms of how these fluctuations are tolerated are needed to be further studied.This thesis consist of three parts. The first part is brain proteome study of hibernating bats Rhinoluphusferrumequinum. Due to the important role of brain during hibernation, we questioned what adaptive changes have evolved in bats’brain during hibernation. We compared the brain proteome of torpid bats and active bats to reveal the changing level of proteins in brain and understand the potential mechanism of brain protection. Results showed that over 36%(36 proteins) of identified proteins with significant expression changes were involved in the synaptic vesicle dynamics and the integrity of cytoskeletons, suggesting the importance of structural foundation in neural activities. In addition, the proteins related to neural mitochondria function (13 proteins) and energy metabolism (24 proteins) were also differentially expressed in torpid bats. These protein changes represent, at least in part, the adaptive response to restricted energy supply in maintenance of neural functions during hibernation. Lots of proteins were also identified with significant changes were closely associated with the regulation of cellular homeostasis, including transcription and translation (6 proteins), proteostasis (10 proteins), and redox homeostasis (9 proteins). Our study describes and reveals the adjustments of brain proteins involving in maintenance of cerebral functions in bats, including structure integrity, energy metabolism, and cellular homeostasis. In addition, GNAO1 may play an important role in maintaining the cerebral activities during hibernation.The second part of this thesis is a comprehensive study of antioxidant systems in hibernating bats’brain. Since bats can tolerate the oxygen fluctuations which are lethal to non-hibernators, the bats may have strategies to deal with oxidative stress and protect themselves from injuries. Until now, comprehensive studies of antioxidant systems in hibernating bats are rare, and whether the reactive oxygen species (ROS) are accumulated in brain of hibernating bats remains unknown. Therefore, we investigated the ROS levels in hibernating bats and outgroup species, and examined the amounts of antioxidant enzymes and low molecular weight antioxidants (LMWAs) in different hibernation states of bats. We found that the total level ROS and RNS in the brain of each of the two distantly related hibernating bats Myotis ricketti and Rhinolophus ferrumequinum at arousal was lower than that at torpid or active state. Different bat species use different antioxidant strategies to deal with the same oxidative stresses. M. ricketti bats upregulated the expression of some enzymes to overcome oxidative stresses, R. ferrumequinum bats maintained a relatively high level some antioxidant enzymes throughout the three different states of hibernation cycles but rapidly upregulated the levels of glutathione (GSH) after torpor. Furthermore, we use Ingenuity pathway analysis tool to predict the transcription factors that may regulate these antioxidant processes. Nrf2 and FOXOs play major roles in the regulation of antioxidant defenses in the brains of bats during hibernation. Our study revealed strategies used by bats against oxidative stresses during hibernation and provide us a new sight to understand the relationship between oxidative stress and antioxidant defense.The third part of this thesis is the evolution of antioxidant related gene. Frugivorous bats eat large amounts of fruits that contain high levels of LMWAs, thus, a reliance on LMWAs might greatly reduce the need for antioxidant enzymes in comparison to insectivorous bats. It is possible that frugivorous bats have a reduced need for Nrf2 function that plays an important role in regulating the transcription of antioxidant enzymes. We thus speculate the Nrf2 gene has undergone relaxed evolution in fruit-eating bats. We obtained Nrf2 sequences from 11 species of bats using molecular cloning methods. The results of molecular evolutionary analyses revealed changes in the selection pressure acting on Nrf2 gene and identified seven specific amino acid substitutions that occurred on the ancestral lineage leading to Old World fruit bats, and also suggested that Nrf2 gene might have experienced relaxed constraint in Old World fruit bats, however, we cannot rule out the possibility of positive selection. Biochemical experiments were also conducted to examine the expression of Nrf2 in Old World fruit bats, results showed that the amount of catalase, which is regulated by Nrf2, was significantly lower in Old World fruit bats despite higher levels of Nrf2 protein in Old World fruit bats. Therefore, the accumulated amino acid substitutions might affect the transcriptional activity in Old World fruit bats. Our study provides the evidence on how the dietary intake influences the antioxidant defense in mammals.In conclusion, brain proteomes of torpid and active bats are compared and results showed that brain functions, including synaptic dynamics and cytoskeleton, energy metabolism, and cellular homeostasis are largely changed during hibernation. A comprehensive study on antioxidant defense of brain in hibernating bats and revealed a specie-specific strategy of antioxidant defense. Combined with the specificity of the feeding habits in bats, we provide the evidence on the evolutionary history of chiropteran feeding habits and antioxidant defenses. These data not only provide us many information on bats hibernation, but also greatly benefit human health research, particularly for development of therapies for brain diseases.