Spatially resolved multiomics on the neuronal effects induced by spaceflight in mice

Long-duration space missions pose significant health risks to astronauts, including central nervous system (CNS) impairment. Exposure to the space environment, including galactic cosmic radiation and microgravity, causes DNA damage, oxidative stress, bone and muscle loss, circadian and sleep dysregulation, microbial dysbiosis, and tissue and organ degeneration. Previous studies on animal models have demonstrated spaceflight stressors impacting brain molecular mechanisms, resulting in neuroinflammation, neurodegeneration, and neurovascular damage. However, a deeper understanding of the mechanisms and specificity of these impairments is crucial for developing effective countermeasures. This study utilizes a novel approach combining single-nucleus multiomics and spatial transcriptomics to investigate spaceflight-induced molecular changes in the mouse brain, addressing the need for detailed, cell-type specific analysis of CNS responses to spaceflight. The use of single-nucleus transcriptomics allows for the analysis of frozen samples, a necessity for spaceflight studies given the limitations of sample processing on-board the International Space Station (ISS). Spatial transcriptomics provides crucial spatial context, enabling the analysis of the impact of spaceflight across different brain regions, complementing the single-cell resolution provided by multiomics analysis. The integration of these two approaches promises a higher resolution analysis than has been possible before and is a crucial first step in understanding the complex effects of spaceflight on the brain.

The literature extensively documents the detrimental effects of spaceflight on various physiological systems. Studies on simulated space radiation reveal neurodegeneration and neuroinflammation in vivo and in vitro, alongside cognitive and behavioral deficits in rodent models. Microgravity in low-Earth orbit leads to fluid redistribution, causing cardiovascular and CNS changes. Previous studies using rodent models exposed to spaceflight have shown various levels of neuroinflammation, neurodegeneration, and neurovascular damage; however, a comprehensive cellular and spatial understanding of these changes has been lacking. Single-cell sequencing methods are powerful tools for the detailed study of individual cell types, but lack spatial context. Spatial transcriptomics, however, provides the complementary spatial component that allows for the investigation of the impact of spaceflight across different brain regions. The integration of these methodologies promises detailed insights into the impact of spaceflight on the nervous system at a level of resolution that is crucial for understanding this complex physiological challenge.

This study utilized brain samples from female BALB/c mice from the Rodent Research-3 (RR-3) mission on the ISS. Three spaceflight mice (F1, F2, F3) and three ground control mice (G1, G2, G3) were used. For each mouse, one brain hemisphere was used for single-nucleus multiomics analysis (snMultiomics; RNA-seq and ATAC-seq), and the other hemisphere for spatial transcriptomics (ST) analysis focusing on the hippocampus. RNA integrity was verified (average RIN 9.15). For snMultiomics, 21,178 nuclei were analyzed, yielding RNA expression profiles and chromatin accessibility information from the same nuclei. Joint clustering analysis identified 18 snMultiomics clusters, categorized into 11 functional groups based on marker genes. For ST, two coronal sections from each hemisphere were analyzed, yielding 29,770 spots. Unsupervised clustering identified 18 distinct spatial clusters. Differential expression analysis identified differentially expressed genes (DEGs) between spaceflight and ground control samples in both snMultiomics and ST datasets. Ligand-receptor interaction analysis was performed using CellPhoneDB and SpatialDM, investigating interactions between cell types affected by spaceflight. Motif analysis using ChromVAR was performed on snATAC-seq data to identify spaceflight-mediated differences in transcription factor activity. Spatial pattern analysis using MISTY assessed signaling pathway changes in relation to cell type colocalization. Metabolic pathway analysis was conducted using GSEA, and smFISH validated gene expression changes. All data were made publicly available through NASA GeneLab and an interactive data portal.

Single-nucleus multiomics analysis revealed 825 differentially expressed genes (DEGs) between spaceflight and ground control samples. These DEGs were primarily involved in neuronal development, axonal/dendritic outgrowth, and synaptic transmission. Comparison with bulk RNA-seq data from the same mice showed that the majority of spaceflight effects are cell-type specific. Comparison with other NASA datasets revealed 461 overlapping DEGs. Spatial transcriptomics analysis identified 4057 DEGs in seven spatial clusters. The most significant changes were observed in cortical neurons (bottom layers), with DEGs related to neuronal development and synaptic transmission. Pathway analysis highlighted neurodegeneration-associated pathways, including protein misfolding. Integration of multiomics and ST data revealed that spaceflight impacted synaptic transmission in the cortex (neurons and astrocytes) and dopaminergic neuron development in the striatum. Ligand-receptor interaction analysis showed upregulation of interactions involved in CNS development. Motif analysis showed spaceflight-mediated changes in transcription factor activity, including reduced neuronal differentiation and increased inflammatory responses. Spatial gene expression pattern analysis revealed spaceflight-associated changes in signaling pathways, including decreased MAPK signaling in neurovasculature and changes in the local neighborhood of other cell types. Metabolic pathway analysis revealed decreased oxidative phosphorylation, glycolysis/gluconeogenesis, fructose/mannose metabolism, and arachidonic acid metabolism, consistent with mitochondrial impairments and astrocyte dysfunction. smFISH validated the upregulation of Adcyl and Gpc5 in spaceflight samples.

This study provides high-resolution insights into the effects of spaceflight on the CNS, revealing alterations in neurogenesis, synaptogenesis, and neuronal development, alongside neurodegeneration and inflammation. The findings show similarities to terrestrial neurodegenerative diseases and aging, suggesting that spaceflight might serve as an accelerated aging model. The study highlights the complementary nature of spatial transcriptomics and single-nucleus multiomics, revealing both regional and cell-type specific effects. The identified changes in circadian gene expression are consistent with circadian disruption, a major biological response to spaceflight. The observed alterations in striatal gene expression and transcription factor accessibility warrant further investigation due to their potential impact on dopaminergic signaling. This study has implications for the development of countermeasures for spaceflight-induced CNS impairment.

This study demonstrates the power of integrating spatial transcriptomics and single-cell multiomics to investigate the complex effects of spaceflight on the CNS. The findings reveal significant alterations in various neuronal processes, showing similarities to neurodegenerative diseases and aging. The results highlight the need for further research to validate these findings with larger sample sizes, to address the limitations of the sample size and mouse sex and to investigate the long-term effects of spaceflight on CNS health. Data sharing fosters collaboration and accelerates the understanding of spaceflight's impact on biological systems.

The study has two main limitations: (1) The limited sample size of legacy samples from the RR-3 mission affects the statistical robustness and limits the interpretability of spatial and multiomics cluster differences. Future studies with larger sample sizes, and ideally including a reacclimation phase, are needed for confirmation. (2) The focus on female mice limits the generalizability of the findings to human astronauts, who comprise both males and females. Future studies should include male mice to address sex dimorphism.