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

Impairment of the central nervous system (CNS) poses a significant health risk for astronauts during long-duration space missions. In this study, we employed an innovative approach by integrating single-cell multiomics (transcriptomics and chromatin accessibility) with spatial transcriptomics to elucidate the impact of spaceflight on the mouse brain in female mice. Our comparative analysis between ground control and spaceflight-exposed animals revealed significant alterations in essential brain processes including neurogenesis, synaptogenesis and synaptic transmission, particularly affecting the cortex, hippocampus, striatum and neuroendocrine structures. Additionally, we observed astrocyte activation and signs of immune dysfunction. At the pathway level, some spaceflight-induced changes in the brain exhibit similarities with neurodegenerative disorders, marked by oxidative stress and protein misfolding. Our integrated spatial multiomics approach serves as a stepping stone towards understanding spaceflight-induced CNS impairments at the level of individual brain regions and cell types, and provides a basis for comparison in future spaceflight studies. For broader scientific impact, all datasets from this study are available through an interactive data portal, as well as the National Aeronautics and Space Administration (NASA) Open Science Data Repository (OSDR).

Cosmic kidney disease: an integrated pan-omic, physiological and morphological study into spaceflight-induced renal dysfunction.

Missions into Deep Space are planned this decade. Yet the health consequences of exposure to microgravity and galactic cosmic radiation (GCR) over years-long missions on indispensable visceral organs such as the kidney are largely unexplored. We performed biomolecular (epigenomic, transcriptomic, proteomic, epiproteomic, metabolomic, metagenomic), clinical chemistry (electrolytes, endocrinology, biochemistry) and morphometry (histology, 3D imaging, miRNA-ISH, tissue weights) analyses using samples and datasets available from 11 spaceflight-exposed mouse and 5 human, 1 simulated microgravity rat and 4 simulated GCR-exposed mouse missions. We found that spaceflight induces: 1) renal transporter dephosphorylation which may indicate astronauts' increased risk of nephrolithiasis is in part a primary renal phenomenon rather than solely a secondary consequence of bone loss; 2) remodelling of the nephron that results in expansion of distal convoluted tubule size but loss of overall tubule density; 3) renal damage and dysfunction when exposed to a Mars roundtrip dose-equivalent of simulated GCR.

NASA GeneLab RNA-seq consensus pipeline: standardized processing of short-read RNA-seq data.

With the development of transcriptomic technologies, we are able to quantify precise changes in gene expression profiles from astronauts and other organisms exposed to spaceflight. Members of NASA GeneLab and GeneLab-associated analysis working groups (AWGs) have developed a consensus pipeline for analyzing short-read RNA-sequencing data from spaceflight-associated experiments. The pipeline includes quality control, read trimming, mapping, and gene quantification steps, culminating in the detection of differentially expressed genes. This data analysis pipeline and the results of its execution using data submitted to GeneLab are now all publicly available through the GeneLab database. We present here the full details and rationale for the construction of this pipeline in order to promote transparency, reproducibility, and reusability of pipeline data; to provide a template for data processing of future spaceflight-relevant datasets; and to encourage cross-analysis of data from other databases with the data available in GeneLab.

NASA GeneLab: interfaces for the exploration of space omics data.

The mission of NASA's GeneLab database (https://genelab.nasa.gov/) is to collect, curate, and provide access to the genomic, transcriptomic, proteomic and metabolomic (so-called 'omics') data from biospecimens flown in space or exposed to simulated space stressors, maximizing their utilization. This large collection of data enables the exploration of molecular network responses to space environments using a systems biology approach. We review here the various components of the GeneLab platform, including the new data repository web interface, and the GeneLab Online Data Entry (GEODE) web portal, which will support the expansion of the database in the future to include companion non-omics assay data. We discuss our design for GEODE, particularly how it promotes investigators providing more accurate metadata, reducing the curation effort required of GeneLab staff. We also introduce here a new GeneLab Application Programming Interface (API) specifically designed to support tools for the visualization of processed omics data. We review the outreach efforts by GeneLab to utilize the spaceflight data in the repository to generate novel discoveries and develop new hypotheses, including spearheading data analysis working groups, and a high school student training program. All these efforts are aimed ultimately at supporting precision risk management for human space exploration.

A New Era for Space Life Science: International Standards for Space Omics Processing.

Space agencies have announced plans for human missions to the Moon to prepare for Mars. However, the space environment presents stressors that include radiation, microgravity, and isolation. Understanding how these factors affect biology is crucial for safe and effective crewed space exploration. There is a need to develop countermeasures, to adapt plants and microbes for nutrient sources and bioregenerative life support, and to limit pathogen infection. Scientists across the world are conducting space omics experiments on model organisms and, more recently, on humans. Optimal extraction of actionable scientific discoveries from these precious datasets will only occur at the collective level with improved standardization. To address this shortcoming, we established ISSOP (International Standards for Space Omics Processing), an international consortium of scientists who aim to enhance standard guidelines between space biologists at a global level. Here we introduce our consortium and share past lessons learned and future challenges related to spaceflight omics.

Advancing the Integration of Biosciences Data Sharing to Further Enable Space Exploration.

Understanding the impact of space exploration remains biologically elusive. Cell Press is dedicating this month to spaceflight (Afshinnekoo et al., 2020), with the open science NASA GeneLab database enabling the study revealing mitochondria as a key biological feature from spaceflight (da Silveira et al., 2020).

Exploring the Effects of Spaceflight on Mouse Physiology using the Open Access NASA GeneLab Platform.

Performing biological experiments in space requires special accommodations and procedures to ensure that these investigations are performed effectively and efficiently. Moreover, given the infrequency of these experiments it is imperative that their impacts be maximized. The rapid advancement of omics technologies offers an opportunity to dramatically increase the volume of data produced from precious spaceflight specimens. To capitalize on this, NASA has developed the GeneLab platform to provide unrestricted access to spaceflight omics data and encourage its widespread analysis. Rodents (both rats and mice) are common model organisms used by scientists to investigate space-related biological impacts. The enclosure that house rodents during spaceflight are called Rodent Habitats (formerly Animal Enclosure Modules), and are substantially different from standard vivarium cages in their dimensions, air flow, and access to water and food. In addition, due to environmental and atmospheric conditions on the International Space Station (ISS), animals are exposed to a higher CO2 concentration. We recently reported that mice in the Rodent Habitats experience large changes in their transcriptome irrespective of whether animals were on the ground or in space. Furthermore, these changes were consistent with a hypoxic response, potentially driven by higher CO2 concentrations. Here we describe how a typical rodent experiment is performed in space, how omics data from these experiments can be accessed through the GeneLab platform, and how to identify key factors in this data. Using this process, any individual can make critical discoveries that could change the design of future space missions and activities.

GeneLab: Omics database for spaceflight experiments.

MOTIVATION: To curate and organize expensive spaceflight experiments conducted aboard space stations and maximize the scientific return of investment, while democratizing access to vast amounts of spaceflight related omics data generated from several model organisms. RESULTS: The GeneLab Data System (GLDS) is an open access database containing fully coordinated and curated 'omics' (genomics, transcriptomics, proteomics, metabolomics) data, detailed metadata and radiation dosimetry for a variety of model organisms. GLDS is supported by an integrated data system allowing federated search across several public bioinformatics repositories. Archived datasets can be queried using full-text search (e.g. keywords, Boolean and wildcards) and results can be sorted in multifactorial manner using assistive filters. GLDS also provides a collaborative platform built on GenomeSpace for sharing files and analyses with collaborators. It currently houses 172 datasets and supports standard guidelines for submission of datasets, MIAME (for microarray), ENCODE Consortium Guidelines (for RNA-seq) and MIAPE Guidelines (for proteomics). AVAILABILITY AND IMPLEMENTATION: https://genelab.nasa.gov/.

NASA GeneLab Project: Bridging Space Radiation Omics with Ground Studies.

Accurate assessment of risks of long-term space missions is critical for human space exploration. It is essential to have a detailed understanding of the biological effects on humans living and working in deep space. Ionizing radiation from galactic cosmic rays (GCR) is a major health risk factor for astronauts on extended missions outside the protective effects of the Earth's magnetic field. Currently, there are gaps in our knowledge of the health risks associated with chronic low-dose, low-dose-rate ionizing radiation, specifically ions associated with high (H) atomic number (Z) and energy (E). The NASA GeneLab project ( https://genelab.nasa.gov/ ) aims to provide a detailed library of omics datasets associated with biological samples exposed to HZE. The GeneLab Data System (GLDS) includes datasets from both spaceflight and ground-based studies, a majority of which involve exposure to ionizing radiation. In addition to detailed information on radiation exposure for ground-based studies, GeneLab is adding detailed, curated dosimetry information for spaceflight experiments. GeneLab is the first comprehensive omics database for space-related research from which an investigator can generate hypotheses to direct future experiments, utilizing both ground and space biological radiation data. The GLDS is continually expanding as omics-related data are generated by the space life sciences community. Here we provide a brief summary of the space radiation-related data available at GeneLab.

Pathogenesis of severe ataxia and tremor without the typical signs of neurodegeneration.

Neurological diseases are especially devastating when they involve neurodegeneration. Neuronal destruction is widespread in cognitive disorders such as Alzheimer's and regionally localized in motor disorders such as Parkinson's, Huntington's, and ataxia. But, surprisingly, the onset and progression of these diseases can occur without neurodegeneration. To understand the origins of diseases that do not have an obvious neuropathology, we tested how loss of CAR8, a regulator of IP3R1-mediated Ca(2+)-signaling, influences cerebellar circuit formation and neural function as movement deteriorates. We found that faulty molecular patterning, which shapes functional circuits called zones, leads to alterations in cerebellar wiring and Purkinje cell activity, but not to degeneration. Rescuing Purkinje cell function improved movement and reducing their Ca(2+) influx eliminated ectopic zones. Our findings in Car8(wdl) mutant mice unveil a pathophysiological mechanism that may operate broadly to impact motor and non-motor conditions that do not involve degeneration.

Parasagittal compartmentation of cerebellar mossy fibers as revealed by the patterned expression of vesicular glutamate transporters VGLUT1 and VGLUT2.

The cerebellum receives sensory signals from spinocerebellar (lower limbs) and dorsal column nuclei (upper limbs) mossy fibers. In the cerebellum, mossy fibers terminate in bands that are topographically aligned with stripes of Purkinje cells. While much is known about the molecular heterogeneity of Purkinje cell stripes, little is known about whether mossy fiber compartments have distinct molecular profiles. Here, we show that the vesicular glutamate transporters VGLUT1 and VGLUT2, which mediate glutamate uptake into synaptic vesicles of excitatory neurons, are expressed in complementary bands of mossy fibers in the adult mouse cerebellum. Using a combination of immunohistochemistry and anterograde tracing, we found heavy VGLUT2 and weak VGLUT1 expression in bands of spinocerebellar mossy fibers. The adjacent bands, which are in part comprised of dorsal column nuclei mossy fibers, strongly express VGLUT1 and weakly express VGLUT2. Simultaneous injections of fluorescent tracers into the dorsal column nuclei and lower thoracic-upper lumbar spinal cord revealed that upper and lower limb sensory pathways innervate adjacent VGLUT1/VGLUT2 parasagittal bands. In summary, we demonstrate that VGLUT1 and VGLUT2 are differentially expressed by dorsal column nuclei and spinocerebellar mossy fibers, which project to complementary cerebellar bands and respect common compartmental boundaries in the adult mouse cerebellum.

Revealing neural circuit topography in multi-color.

Neural circuits are organized into functional topographic maps. In order to visualize complex circuit architecture we developed an approach to reliably label the global patterning of multiple topographic projections. The cerebellum is an ideal model to study the orderly arrangement of neural circuits. For example, the compartmental organization of spinocerebellar mossy fibers has proven to be an indispensable system for studying mossy fiber patterning. We recently showed that wheat germ agglutinin (WGA) conjugated to Alexa 555 and 488 can be used for tracing spinocerebellar mossy fiber projections in developing and adult mice (Reeber et al. 2011). We found three major properties that make the WGA-Alexa tracers desirable tools for labeling neural projections. First, Alexa fluorophores are intense and their brightness allows for wholemount imaging directly after tracing. Second, WGA-Alexa tracers label the entire trajectory of developing and adult neural projections. Third, WGA-Alexa tracers are rapidly transported in both retrograde and anterograde directions. Here, we describe in detail how to prepare the tracers and other required tools, how to perform the surgery for spinocerebellar tracing and how best to image traced projections in three dimensions. In summary, we provide a step-by-step tracing protocol that will be useful for deciphering the organization and connectivity of functional maps not only in the cerebellum but also in the cortex, brainstem, and spinal cord.

Fluorescence mapping of afferent topography in three dimensions.

Neural circuits are organized into complex topographic maps. Although several neuroanatomical and genetic tools are available for studying circuit architecture, a limited number of methods exist for reliably revealing the global patterning of multiple topographic projections. Here we used wheat germ agglutinin (WGA) conjugated to Alexa 555 and 488 for dual color fluorescent mapping of parasagittal spinocerebellar topography in three dimensions. Using tissue section and wholemount imaging we show that WGA-Alexa tracers have three main characteristics that make them ideal tools for analyses of neural projection topography. First, the intense brightness of Alexa fluorophores allows multi-color imaging of patterned afferent projections in wholemount preparations. Second, WGA-Alexa tracers robustly label the entire trajectory of developing and adult projections. Third, long tracts such as the adult spinocerebellar tract can be traced in less than 6 h. Moreover, using WGA-Alexa tracers we resolved a level of complexity in the compartmentalized topography of the spinocerebellar projection map that has never before been appreciated. In summary, we introduce versatile tracers for rapidly labeling multiple topographic projections in three dimensions and uncover wiring complexities in the spinocerebellar map.

Neurofilament heavy chain expression reveals a unique parasagittal stripe topography in the mouse cerebellum.

Despite the general uniformity in cellular composition of the adult cerebellum (Cb), the expression of proteins such as ZebrinII/AldolaseC and the small heat shock protein HSP25 reveal striking patterns of parasagittal Purkinje cell (PC) stripes. Based on differences in the stripe configuration within subsets of lobules, the Cb can be further divided into four anterior-posterior transverse zones: anterior zone (AZ) = lobules I-V, central zone (CZ) = lobules VI-VII, posterior zone (PZ) = lobules VIII and anterior IX, and the nodular zone (NZ) = lobules posterior IX-X. Here we used whole-mount and tissue section immunohistochemistry to show that neurofilament heavy chain (NFH) expression alone divides all lobules of the mouse Cb into a complex series of parasagittal stripes of PCs. We revealed that the striped pattern of NFH in the vermis of the AZ and PZ was complementary to ZebrinII and phospholipase C ß3 (PLCß3), and corresponded to phospholipase C ß4 (PLCß4). In the CZ and NZ the stripe pattern of NFH was complementary to HSP25 and corresponded to PLCß3. The boundaries of the NFH stripes were not always sharply delineated. Instead, a gradual decrease in NFH expression was observed toward the edges of particular stripes, resulting in domains comprised of overlapping expression patterns. Furthermore, the terminal field distributions of mossy and climbing fibers had a complex but consistent topographical alignment with NFH stripes. In summary, NFH expression reveals an exquisite level of Cb stripe complexity that respects the transverse zone divisions and delineates an intricately patterned target field for Cb afferents.