Gene Expression Profile Changes In The Vestibular Structures Of Mice After A 30-day Space Flight

Projects related to the launch of animals into space are always resource-intensive,and the results obtained during their implementation are unique,so,can act as a control for comparing and verifying data obtained in terrestrial conditions.The inferior olive nuclei and medial vestibular nuclei have been found to be significantly activated in space,but almost nothing is known about the molecular mechanisms that underlie the ability for neuroplasticity in microgravity in these structures.The aim of this research is to study gene expression profile changes in the medial vestibular nuclei and the inferior olive nuclei of the brain of mice after a 30-day space flight on the Bion-M1 satellite and compare the gene expression profile with the data obtained from mice exposed to the simulated microgravity in the model of hindlimb unloading.In April 2013,the Bion-M1 spacecraft was launched into near-earth orbit,with laboratory mice(mus musculus L)of the C57BL6 line on board(male,4-month age).On May 19-20,2013,the animals successfully returned to Earth after a 30-day flight,mice were sacrificed by the method of cervical dislocation.Isolation of structures VEvestibular nuclei area,IO-inferior olives area from the living brain occurred on ice in several minutes.Also,samples from the control group kept for 30 days on Earth under conditions that simulate the habitat on a biosatellite were collected.The construction of cDNA libraries was performed and genome-wide gene expression profiling by the RNA-Seq method on the Illumina Hi Seq2500 platform was carried out.For further analysis,7 VE transcriptomes(4 space,3 controls)and 6 IO transcriptomes(4 space,2 controls)were used.Bioinformatics analysis was performed.The readings obtained were correlated with the mouse genome(Gene Cod v.27 version;STAR).Statistical analysis was performed in R language(DESeq2,ggplot2,PCA,MCR,Metascape service)Comparison groups were created to study the difference between gene expression in structures from space and control samples,compared gene expression profile from olive and vestibular nuclei with the basic expression of brain genes.Hindlimb unloading microgravity simulation model in the research was based on the effect of the fluid shift.Material from four control and six experimental C57BL6 line mice(male,4-month age)was analyzed.Droplet Digital Polymerase Chain Reaction(dd PCR)was used to test the relevance of this widely used model to confirm or reject the bioinformatic results from the real spaceflight.The data obtained from dd PCR were analyzed with “RStudio” program(version1.4.1106,RStudio,PBC)and “Past”(V.4.03).In case of limited number of samples non-parametric methods were used(Mann-Whitney).After a 30-day space flight on the BION-M1 satellite,an increase in differentially expressed genes associated with angiogenesis,growth factors and extracellular matrix changes was observed in the Inferior Olive Nuclei and an increase in genes associated with mitochondrial responses was observed in the Medial Vestibular Nuclei of C57BL6 mice.A decrease in the expression of genes associated with the negative regulation of catabolic processes and mRNA processing was revealed in IO area and in VE area a decrease in the expression of genes associated with lipid metabolism,intracellular transport,and transport of neurotransmitters was found.Differential expression of genes MPZ,MEG3,Plxnb3,Unc13 a,Junb(IO)and PLVAP,Pou3f2,ERC1,ND1,COX1(VE)was confirmed by the dd PCR.The Plxnb3,Unc13 a,Junb,PLVAP,ND1 and COX1 genes revealed a change in the expression profile similar to the results obtained using bioinformatic analysis of sequencing data.Simulation of microgravity based on the hind limb unloading model has shown the relevance of this model in relation to changes in the gene expression profile in the Medial Vestibular Nuclei associated with the PLVAP and ND1 genesMyelin Protein Zero(MPZ)gene tended to show the most significant fold change in bioinformatic analysis in IO area after a 30-day space flight.The data obtained contradict the known literature data.Thus,the effect of a real space flight can actually increase the expression of this gene only in the IO zone and decrease its expression in other parts of the CNS.Thus,this gene requires further research in relation to expression in the vestibular structures of the mouse brain.