Evaluation of Gene, Protein and Neurotrophin Expression in the Brain of Mice Exposed to Space Environment for 91 Days

Protein and Neurotrophin Expression in the Brain of Mice Exposed to Space Environment for 91 Days. PLoS ONE 7(7): e40112. doi:10.1371/journal.pone.0040112 Evaluation of Gene, Protein and Neurotrophin Expression in the Brain of Mice Exposed to Space Environment for 91 Days Daniela Santucci 0 Fuminori Kawano 0 Takashi Ohira 0 Masahiro Terada 0 Naoya Nakai 0 Nadia Francia 0 Enrico Alleva 0 Luigi Aloe 0 Toshimasa Ochiai 0 Ranieri Cancedda 0 Katsumasa Goto 0 Yoshinobu Ohira 0 Jeffrey M. Gimble, Pennington Biomedical Research Center, United States of America 0 1 Behavioural Neuroscience Section, Cellular Biology and Neuroscience Department, Istituto Superiore di Sanita` , Rome , Italy , 2 Graduate School of Medicine, Osaka University , Osaka , Japan , 3 Graduate School of Frontier Biosciences, Osaka University , Osaka, Japan, 4 Japan Aerospace Exploration Agency, Ibaraki , Japan , 5 Institute of Neurobiology and Molecular Medicine, CNR, European Brain Research Institute , Rome, Italy, 6 Mitsubishi Heavy Industries, Hyogo, Japan, 7 DOBIG , University of Genova , Genova , Italy , 8 Graduate School of Health Sciences, Toyohashi SOZO University , Aichi , Japan Effects of 3-month exposure to microgravity environment on the expression of genes and proteins in mouse brain were studied. Moreover, responses of neurobiological parameters, nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF), were also evaluated in the cerebellum, hippocampus, cortex, and adrenal glands. Spaceflight-related changes in gene and protein expression were observed. Biological processes of the up-regulated genes were related to the immune response, metabolic process, and/or inflammatory response. Changes of cellular components involving in microsome and vesicular fraction were also noted. Molecular function categories were related to various enzyme activities. The biological processes in the down-regulated genes were related to various metabolic and catabolic processes. Cellular components were related to cytoplasm and mitochondrion. The down-regulated molecular functions were related to catalytic and oxidoreductase activities. Up-regulation of 28 proteins was seen following spaceflight vs. those in ground control. These proteins were related to mitochondrial metabolism, synthesis and hydrolysis of ATP, calcium/calmodulin metabolism, nervous system, and transport of proteins and/or amino acids. Down-regulated proteins were related to mitochondrial metabolism. Expression of NGF in hippocampus, cortex, and adrenal gland of wild type animal tended to decrease following spaceflight. As for pleiotrophin transgenic mice, spaceflight-related reduction of NGF occured only in adrenal gland. Consistent trends between various portions of brain and adrenal gland were not observed in the responses of BDNF to spaceflight. Although exposure to real microgravity influenced the expression of a number of genes and proteins in the brain that have been shown to be involved in a wide spectrum of biological function, it is still unclear how the functional properties of brain were influenced by 3-month exposure to microgravity. - Altered gravitational environment represents a unique challenge for biological systems that have evolved against a constant gravitational background and it has been reported that exposure to actual and/or simulated microgravity, as well as to rotationally induced hypergravity, causes various physiological adaptations, including in the central nervous system (CNS) [14] and antigravity muscle [5,6] in mice, rats and humans. For example, 16 days of spaceflight caused a change in the synaptic circuitry at the hindlimb cortex of the postnatal developing rats [2], while exposure of rats to hypergravity from day 11 of gestation to postnatal day 15 led to substantial and persistent delay in the development of the cortical monaminergic projections to the spinal cord in young rats [7] or to altered cerebellar growth [8]. In addition, rats flown on the space shuttle (Neurolab) from postnatal day 8 to 24 in 1998 showed an abnormal development of extensor motoneurons and changes in the number and morphology of cortical synapses [9]. Analogously, exposure to hypergravity affects exploratory behavior and ability to discriminate a new spatial arrangement [10,11], as well as nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF) levels, in the CNS of mice [12,13]. Moreover, modulation of genes coding for proteins involved in a wide range of cellular functions (DNA/RNA metabolism, protein processing, intermediate metabolism, cytoskeleton and motility, cell cycle and apoptosis, signal transduction, and neuronal structure/function) has been seen in the brain of adult mice exposed to acute 2-G hypergravity [14]. In particular, in terms of the nervous system, the hindlimb suspension, which is often used as the simulation model for exposure to microgravity environment, causes the decrease of electromyogram activity in soleus muscle, afferent neurograms in spinal cord [15], GABAergic neurons in the hindlimb somatosensory cortex [1], and neurogenesis in rats [16]. Recently, proteomic analyses of the hippocampus [17] and the hypothalamus [18], as well as a microarray analysis of gene expression in mouse brain [19], were performed to elucidate the mechanism responsible for the adaptation to gravitational unloading. After 7 days of hindlimb suspension, the expression of cytoskeletal proteins, such as tubulin and metabolic proteins in hippocampus of adult mice changed. Seven spots were decreased and 4 spots were increased [17], and biomarkers of oxidative stress in hypothalamus of mice were increased [18]. Therefore, exposure to simulated microgravity environments by hindlimb suspension might also induce distinct changes specific to the regions of the brain. However, it is not clear how the characteristics of mouse brain respond to long-term inhibition of antigravity activity. Therefore, the current study was performed to investigate the effects of longterm exposure to microgravity environment on the characteristics of brain in mice, since we had the access to brain samples by participating in the tissue sharing team [20]. Specifically, comprehensive analyses of gene and protein expression were performed. Further, responses of NGF and BDNF in brain, as well as adrenal glands where the level of neutrophin expression is also regulated [21], were investigated, because the changes in neurotrophin levels in CNS following exposure to challenging environment and the roles of these neurobiological determinants were also reported [22]. Materials and Methods Experimental Design and Animal Care The protocol utilized in the study has been authorized by the Public Veterinary Health Department of the Italian Ministry of Health. The experimental procedures were also conducted in accordance with the Guide for the Care and Use of Laboratory Animals of the Japanese Physiological Society, NIH Guide for the C (...truncated)