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NASA NTRS2015

2015 Space Radiation Standing Review Panel

The 2015 Space Radiation Standing Review Panel (from here on referred to as the SRP) met for a site visit in Houston, TX on December 8 - 9, 2015. The SRP met with representatives from the Space Radiation Element and members of the Human Research Program (HRP) to review the updated research plan for the Risk of Radiation Carcinogenesis Cancer Risk. The SRP also reviewed the newly revised Evidence Reports for the Risk of Acute Radiation Syndromes Due to Solar Particle Events (SPEs) (Acute Risk), the Risk of Acute (In-flight) and Late Central Nervous System Effects from Radiation Exposure (CNS Risk), and the Risk of Cardiovascular Disease and Other Degenerative Tissue Effects from Radiation (Degen Risk), as well as a status update on these Risks. The SRP would like to commend Dr. Simonsen, Dr. Huff, Dr. Nelson, and Dr. Patel for their detailed presentations. The Space Radiation Element did a great job presenting a very large volume of material. The SRP considers it to be a strong program that is well-organized, well-coordinated and generates valuable data. The SRP commended the tissue sharing protocols, working groups, systems biology analysis, and standardization of models. In several of the discussed areas the SRP suggested improvements of the research plans in the future. These include the following: It is important that the team has expanded efforts examining immunology and inflammation as important components of the space radiation biological response. This is an overarching and important focus that is likely to apply to all aspects of the program including acute, CVD, CNS, cancer and others. Given that the area of immunology/inflammation is highly complex (and especially so as it relates to radiation), it warrants the expansion of investigators expertise in immunology and inflammation to work with the individual research projects and also the NASA Specialized Center of Research (NSCORs). Historical data on radiation injury to be entered into the Watson “big data” study must be used with caution. The general scientific issues of reproducibility, details of experimental methods and data analysis from preclinical and basic research laboratories have been raised broadly over the last few years (not specific to this work) and indicate that caution must be applied in the ways these data are used. This pertains to preclinical data and also to phase 3 clinical trials in radiation oncology and medical oncology. Of course, appropriate use and analysis of these “big-data” sets also offer the potential of pinpointing limitations and extracting remaining useful information. Emphasis should be placed on the latter possibility. A key target is risk reduction from radiation exposure. Progress of the entire space program, now moving towards the Mars mission, requires timely answers to key components of human risk, which are known to be complex. Periodic review of progress should be conducted with additional resources directed into achieving critical milestones. Turning the long red bars to yellow and green (or for some risks such as CNS possibly to grey) must be high priority. That such progress will require new science and not engineering means that it should be viewed in a knowledge-based light. The technology-based aspects of engineering issues are certainly as important, however, science and knowledge-based problems are solved in a different way than engineering. Timelines for engineering are more predictable, while for science, progress can be methodical with occasional major incremental findings that can rapidly change the rate of progress. As opportunities for rapid incremental changes arise, periodic enhancement of investment is strongly recommended to enable such new knowledge to be quickly and efficiently exploited. Collaborations and linkages with National Institute of Allergy and Infectious Diseases (NIAID), the Biomedical Advanced Research and Development Authority (BARDA) and the Department of Defense (DoD) are in place and more are encouraged, where possible, with the radiation injury and medical countermeasure studies. This could include utilizing some of their animal model testing contracts to facilitate obtaining results using common platforms. Such approach will facilitate the comparison of results among laboratories, and will facilitate and accelerate the development of medical countermeasures. It is particularly noteworthy that the NASA Space Radiation Element is reaching out to the Multidisciplinary European Low Dose Initiative (MELODI) platform coordinating low dose radiation risk research, and to other international agencies that are studying low dose radiation effects in an effort to fill the void generated by the cancelation of the Department of Energy (DOE) low dose radiation program. While NASA is working actively with NIAID and BARDA to integrate their relevant findings of radiation mitigator investigations to NASA programs, the committee notes its disappointment that the United States currently lacks a dedicated low dose radiation program with clear mechanistic orientation and aimed at the quantification and mitigation of human radiation risk on Earth. This void gives to the NASA Space Radiation Program Element special societal value, but also makes its overall design more challenging.

Steinberg, Susan↗

NASA NTRS2019

Simulated Microgravity Affects Behavior and Cytokine Expression in the Hippocampus of Adult Mice: Influence of Mitochondrial Reactive Oxygen Species

The effects of microgravity, and social isolation on the CNS are poorly understood. We hypothesize that mitochondrial reactive oxygen species (ROS) play an important role in this process. Since mice are social animals, our lab developed a novel social model of hindlimb unloading (HU), enabling us to determine the effects of both social isolation and simulated microgravity. Responses to 30d of HU were compared in wildtype or transgenic MCAT mice who over-express human catalase in mitochondria. Abundance of 4-Hydroxynonenal, Park7 (a redox-sensitive chaperone and sensor of oxidative stress) and corticosterone were measured by ELISA. Cytokines related to inflammation in the hippocampus and in plasma were analyzed by a protein array. Behavioral data was collected over a 24-hour period.Socially housed HU mice were more active and conducted at least two times more exploratory activities, compared to normally loaded mice. Correlation analysis revealed that specific brain and plasma cytokines correspond with specific behaviors. Simulated microgravity and/or social isolation caused changes in cytokine patterns in the hippocampus and in plasma, with significant interaction effects of HU and genotype in expression levels of five cytokines (out of 35). Interestingly, elevation of these generally pro-inflammatory cytokines by HU in WT mice was mitigated in MCAT mice, suggesting a role for mitochondrial ROS signaling in inflammatory CNS responses to microgravity. Interestingly, socially housed mice had also lower level of 4HNE and higher level of Park7 in the hippocampus compared to singly housed animals. The cytokine responses to social isolation were more extensive in brain vs plasma. Further, there was no overlap in the cytokine repertoire regulated in response to microgravity versus, isolation suggesting divergent mechanisms or downstream signaling. These findings implicate a potentially important role for mitochondrial ROS in CNS responses to the challenges posed both by prolonged missions in space and bedrest on Earth

Guttmann, Linda↗

NASA NTRSDate not supplied

Human placenta-derived stromal cells mitigate select immune responses to simulated weightlessness

Space flight presents unique risks to human health. Microgravity and social isolation are important space flight stressors which have vast impact on multiple systems such as the musculoskeletal, immune and central nervous system (CNS). To explore candidate countermeasures against tissue decrements caused by spaceflight, we performed a pilot study with PLacental-eXpanded stromal (PLX) cells. PLX cells secrete factors that modulate the immune system, but do not trigger host immune responses, enabling treatment in subjects that have intact immune systems. These cells had previous success in treating critical limb ischemia and acute radiation syndrome in murine models. In this study, 4-month-old female mice underwent 30 days of hindlimb unloading (HU) in single housing conditions to simulate long-term weightlessness and isolation. HU and normally ambulating controls were administered with PLX cells or sham-injected with Plasmalyte. We hypothesized that administration of PLX cells will mitigate some of the effects caused by prolonged HU. We found that treatment with PLX cells was well tolerated as indicated by body weights, circulating corticosterone levels and immune organ response to injection of the cells. HU led to the expected decreases in soleus muscle weights and bone loss. We did not observe any protective properties of the PLX cells in the musculoskeletal system but found that the cell treatment partially protected from a subset of HU-induced immune and CNS changes. In the hippocampus, protein levels of 9 of 44 cytokines were downregulated by HU. PLX administration mitigated changes in expression of 8 of these cytokines, including IL-2, IL-6 and MCP-1. To our knowledge, this is the first report on the protective effects of PLX-PAD against HU-induced changes in the cytokine milieu of the CNS. Our findings warrant further studies, which includes testing in actual spaceflight.

Microgravity↗

NASA NTRS2021

Microglia Cells, The Brain Innate Immune System: Friend or Foe?

Microglial cells are the resident immune cells of the Central Nervous System(CNS). Under physiological conditions, microglia constantly surveil their surrounding parenchyma and act as scavenger cells to maintain a healthy environment within the CNS. Following different insults to the CNS, microglia turn into a “reactive” state characterized by the production of inflammatory mediators that promote tissue repair to restore homeostasis. If inflammation is not in check, chronic microglia activation results in damage to the brain and leads to persistent cognitive impairments. Microglia display sex-specific features in adult mice; specifically, microglia from female mice have been found to be less reactive. Exposure to space radiation results in chronic activation of microglia in male but not in female mice. Interestingly, manipulating microglia after exposure to space radiation can prevent the development of cognitive deficits in adult male mice. These discoveries may provide clues in how to protect astronauts’ cognitive functions both during the missions and after return

Serena Paladini Maria↗

NASA NTRS2021

Effects of a 33-Ion Sequential Beam Galactic Cosmic Ray Analog on Male Mouse Behavior and Evaluation of CDDO-EA as a Radiation Countermeasure

In long-term spaceflight, astronauts will face unique cognitive loads and social challenges which will be complicated by communication delays with Earth. It is important to understand the central nervous system (CNS) effects of deep spaceflight and the associated unavoidable exposure to galactic cosmic radiation (GCR). Rodent studies show single- or simple-particle combination exposure alters CNS endpoints, including hippocampal-dependent behavior. An even better Earth-based simulation of GCR is now available, consisting of a 33-beam (33-GCR) exposure. However, the effect of whole-body 33-GCR exposure on rodent behavior is unknown, and no 33-GCR CNS countermeasures have been tested. Here astronaut-age-equivalent (6mo-old) C57BL/6J male mice were exposed to 33-GCR (75cGy, a Mars mission dose). Pre-/during/post-Sham or 33-GCR exposure, mice received a diet containing a ‘vehicle’ formulation alone or with the antioxidant/anti-inflammatory compound CDDO-EA as a potential countermeasure. Behavioral testing beginning 4mo post-irradiation suggested radiation and diet did not affect measures of exploration/anxiety-like behaviors (open field, elevated plus maze) or recognition of a novel object. However, in 3-Chamber Social Interaction (3-CSI), CDDO-EA/33-GCR mice failed to spend more time exploring a holder containing a novel mouse vs. a novel object (empty holder), suggesting sociability deficits. Also, Vehicle/33-GCR and CDDO-EA/Sham mice failed to discriminate between a novel stranger vs. familiarized stranger mouse, suggesting blunted preference for social novelty. CDDO-EA given pre-/ during/post-irradiation did not attenuate the 33-GCR-induced blunting of preference for social novelty. Future elucidation of the mechanisms underlying 33-GCR-induced blunting of preference for social novelty will improve risk analysis for astronauts which may in-turn improve countermeasures.

Space radiation↗

NASA NTRS2021

Effects of A 33-Ion Sequential Beam Galactic Cosmic Ray Analog on Male Mouse Behavior and Evaluation of CDDO-EA as A Radiation Countermeasure

Space radiation↗

NASA NTRSDate not supplied

Neurobiological Outcomes of Mice Exposed to Combined Spaceflight Stressors

As NASA plans for exploration beyond Low-Earth Orbit, it is imperative to understand how deep spaceflight stressors impact human physiology. Deep space exposes crew members to multiple NASA-defined risks, including altered gravity, ionizing radiation, and social isolation. While the effects of individual stressors have been studied, there are still gaps in our understanding of their combined effects. In this study, we utilized the simulated five-ion galactic cosmic radiation (GCR) and isolation to assess the combinatorial effects of space stressors on the central nervous system (CNS) in mice. Our study captures sexually dimorphic responses by quantifying CNS outcomes in crewage-matched male and female mice. We assessed CNS responses in mice exposed to four days of isolation, followed by three different dosage of acute radiation exposure (5 cGy, 15 cGy, and 50 cGy 5-ion GCRsim). Comprehensive hippocampal cytokine biomarker analyses were conducted in both males and females at two weeks post-irradiation (intermediate cohort) and 124 days post-irradiation (delayed cohort). Proteomics on hippocampal lysates were conducted in the delayed cohort. Our results provide evidence for sex-specific differentially regulated hippocampal cytokines and proteomic profiles. Immunohistochemical analysis of the mice brain from intermediate and delayed cohorts is currently underway for multiple protein markers, including microglial activation, astrocytes, and dopaminergic neuronal population, among others. This project closely aligns with NASA’s efforts to characterize sex-specific risks associated with deep space exploration. We anticipate the results from this project will aid in our understanding of spaceflight stressors to ensure crew health and performance.

spaceflight↗

NASA NTRS2024

Long-term effects of acute irradiation and isolation on crew age-matched mice

The future of space missions beyond low-Earth orbit presents exciting opportunities but may come with formidable health challenges. Astronauts face a unique combination of space stressors capable of exerting effects on the central nervous system (CNS) with possible sex-dependent differences. The interaction of these combined spaceflight stressors may induce oxidative stress, thereby altering brain integrity and behavioral performance. Understanding these changes is critical for safeguarding astronaut health and enabling successful exploration. By understanding the physiological responses to the combined effects of ionizing radiation (IR) and social isolation in the brain through immunohistochemistry (IHC), proteomics and cytokine expression analyses, we aim to identify CNS pathways ripe for countermeasure development to mitigate adverse brain changes with future deep space exploration. Here, we study the combined effects of simulated 5-ion galactic cosmic radiation (GCRsim) at acute dosage (5, 15, and 50 cGy) and social isolation on crew age-matched male and female mice at 124 days post-irradiation. We conducted analyses of hippocampal cytokine biomarkers and proteomic profiling on hippocampal lysates. Our findings reveal sex-specific regulation of hippocampal cytokines and proteomic profiles. Ongoing immunohistochemistry findings on the hippocampus will provide qualitative information regarding key protein expression and cellular changes such as microglial activation, blood brain barrier integrity, and neuroinflammation. With future plans for long-duration space missions, such as Mars exploration, understanding CNS changes over extended periods in mice can provide insights into potential long-term health impacts on astronauts. This project will provide us with valuable insights into sex-differences in neurobiological processes and disease mechanisms impacting men and women in space.

space radiation↗

DOE OSTI2026

Single‐Cell Nanodroplet Processing Proteomics Pipeline for Analysis of Human‐Derived Microglia

Single-cell omics tools provide unique insights into heterogeneous cell populations and their responses to stimuli. For example, single-cell RNA sequencing has identified several transcriptionally distinct populations of microglia, which are resident immune cells of the central nervous system (CNS) that are responsive to CNS injury, infection, and neurodegeneration. To date, single-cell studies of microglia have focused on RNA-sequencing or cytometry by time of flight (CyTOF), which provide indirect readouts of protein abundance or quantification of a limited number of targets. Herein, we present a workflow based on FACS-assisted isolation, cryopreservation, and nanodroplet-based processing for single-cell mass spectrometry proteomics analysis of the postmortem human brain cortex-derived microglia. From a single microglial cell, 1039 proteins could be identified on average. As a proof-of-principle, we applied single-cell proteomics for exploring the heterogeneity of brain microglia at the cellular level. This pilot proteomics data partially recapitulates the prior microglia subtypes. Specifically, we determined that mitochondrial proteins, in particular members of NADH dehydrogenase (Complex I), cytochrome b-c1 (Complex III), cytochrome c oxidase (Complex IV), F1-ATPase (Complex V), and Na+/K+-ATPase complex, drive variation across microglia. This pipeline offers the potential for identifying functionally and analytically relevant protein targets for microglia in Alzheimer's disease and other neurological disorders.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Carbon nanospikes have improved sensitivity and antifouling properties for adenosine, hydrogen peroxide, and histamine

Carbon nanospikes (CNSs) are a new nanomaterial that has enhanced surface roughness and surface oxide concentration, increasing the sensitivity for dopamine detection. However, CNS-modified electrodes (CNSMEs) have not been characterized for other neurochemicals, particularly those with higher oxidation potentials. The purpose of this study was to evaluate CNSMEs for the detection of adenosine, hydrogen peroxide (H 2 O 2 ), and histamine. The sensitivity increased with CNSs, and signals at CNSMEs were about 3.3 times higher than CFMEs. Normalizing for surface area differences using background currents, CNSMEs show an increased signal of 4.8 times for adenosine, 1.5 times for H 2 O 2 , and 2 times for histamine. CNSMEs promoted the formation of secondary products for adenosine and histamine, which enables differentiation from other analytes with similar oxidation potentials. Furthermore, CNSs also selectively enhance the sensitivity for adenosine and histamine compared to H 2 O 2 . A scan rate test reveals that adenosine is more adsorption-controlled at CNS electrodes than CFMEs. CNSMEs are antifouling for histamine, with less fouling because the polymers formed after histamine electrooxidation do not adsorb due to an elevated number of edge planes. CNSMEs were useful for detecting each analyte applied in brain slices. Because of the hydrophilic surface compared to CFMEs, CNSMEs also have reduced biofouling when used in tissue. Therefore, CNSMEs are useful for tissue measurements of adenosine, hydrogen peroxide, and histamine with high selectivity and low fouling.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2021

Phospholipase D and phosphatidylinositol-4-phosphate 5-kinase 1 are involved in the regulation of oligodendrocyte morphological differentiation

Highlights: • PLD and PIP5K1 are involved in the regulation of oligodendroglial cell morphological differentiation. • Inhibition of PLD or PIP5K1 decreases differentiation or myelin marker expression. • PLD and PIP5K1 regulate differentiation of oligodendrocyte precursor cells. • Inhibition of PLD or PIP5K1 decreases differentiation marker expression of oligodendrocyte precursor cells. Oligodendroglial cells (oligodendrocytes) differentiate to form the myelin that wraps neuronal axons in the central nervous system (CNS). This myelin sheath supports the propagation of saltatory conduction and protects axons from physical stresses. When oligodendrocytes do not normally differentiate to myelinate axons, their key functions as oligodendrocytes in the CNS are severely impaired. The molecular mechanics that control differentiation still remain to be clarified. Arf6 belongs to the small GTPase family and is known to be a positive regulator of oligodendrocyte differentiation. Here, we show that the phospholipase D (PLD) and phosphatidylinositol-4-phosphate 5-kinase 1 (PIP5K1) molecules, the major effectors of Arf6, are involved in the regulation of oligodendrocyte differentiation. Knockdown of PLD1 or PIP5K type 1γ (PIP5K1C) by their respective specific siRNAs in mouse oligodendroglial FBD-102b cells inhibited morphological differentiation into structures bearing myelin-like processes; this finding is consistent with the concurrent changes in expression of differentiation and myelin marker proteins. Treatment with VU0155069 or UNC3230, specific inhibitors of PLD and PIP5K1, respectively, blunted morphological differentiation and decreased expression of myelin and differentiation marker proteins. Similar results have been obtained in studies using primary oligodendrocytes. These results suggest that the major Arf6 effector molecules PLD and PIP5K1 are among the molecules involved in the regulation of morphological differentiation in oligodendrocytes prior to myelination.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2021

Vinculin is required for neuronal mechanosensing but not for axon outgrowth

Integrin receptors are transmembrane proteins that bind to the extracellular matrix (ECM). In most animal cell types integrins cluster together with adaptor proteins at focal adhesions that sense and respond to external mechanical signals. In the central nervous system (CNS), ECM proteins are sparsely distributed, the tissue is comparatively soft and neurons do not form focal adhesions. Thus, how neurons sense tissue stiffness is currently poorly understood. Here, we found that integrins and the integrin-associated proteins talin and focal adhesion kinase (FAK) are required for the outgrowth of neuronal processes. Vinculin, however, whilst not required for neurite outgrowth was a key regulator of integrin-mediated mechanosensing of neurons. During growth, growth cones of axons of CNS derived cells exerted dynamic stresses of around 10–12 Pa on their environment, and axons grew significantly longer on soft (0.4 kPa) compared to stiff (8 kPa) substrates. Depletion of vinculin blocked this ability of growth cones to distinguish between soft and stiff substrates. These data suggest that vinculin in neurons acts as a key mechanosensor, involved in the regulation of growth cone motility.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2023

Genome editing in the mouse brain with minimally immunogenic Cas9 RNPs

Transient delivery of CRISPR-Cas9 ribonucleoproteins (RNPs) into the central nervous system (CNS) for therapeutic genome editing could avoid limitations of viral vector-based delivery including cargo capacity, immunogenicity, and cost. Here, we tested the ability of cell-penetrant Cas9 RNPs to edit the mouse striatum when introduced using a convection-enhanced delivery system. These transient Cas9 RNPs showed comparable editing of neurons and reduced adaptive immune responses relative to one formulation of Cas9 delivered using AAV serotype 9. The production of ultra-low endotoxin Cas9 protein manufactured at scale further improved innate immunity. We conclude that injection-based delivery of minimally immunogenic CRISPR genome editing RNPs into the CNS provides a valuable alternative to virus-mediated genome editing.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2023

Sequence similarity between SARS-CoV-2 nucleocapsid and multiple sclerosis-associated proteins provides insight into viral neuropathogenesis following infection

Abstract The novel coronavirus SARS-CoV-2 continues to cause death and disease throughout the world, underscoring the necessity of understanding the virus and host immune response. From the start of the pandemic, a prominent pattern of central nervous system (CNS) pathologies, including demyelination, has emerged, suggesting an underlying mechanism of viral mimicry to CNS proteins. We hypothesized that immunodominant epitopes of SARS-CoV-2 share homology with proteins associated with multiple sclerosis (MS). Using PEPMatch, a newly developed bioinformatics package which predicts peptide similarity within specific amino acid mismatching parameters consistent with published MHC binding capacity, we discovered that nucleocapsid protein shares significant overlap with 22 MS-associated proteins, including myelin proteolipid protein (PLP). Further computational evaluation demonstrated that this overlap may have critical implications for T cell responses in MS patients and is likely unique to SARS-CoV-2 among the major human coronaviruses. Our findings substantiate the hypothesis of viral molecular mimicry in the pathogenesis of MS and warrant further experimental exploration.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2023

Small molecules disaggregate alpha-synuclein and prevent seeding from patient brain-derived fibrils

The amyloid aggregation of alpha-synuclein within the brain is associated with the pathogenesis of Parkinson’s disease (PD) and other related synucleinopathies, including multiple system atrophy (MSA). Alpha-synuclein aggregates are a major therapeutic target for treatment of these diseases. We identify two small molecules capable of disassembling preformed alpha-synuclein fibrils. The compounds, termed CNS-11 and CNS-11g, disaggregate recombinant alpha-synuclein fibrils in vitro, prevent the intracellular seeded aggregation of alpha-synuclein fibrils, and mitigate alpha-synuclein fibril cytotoxicity in neuronal cells. Furthermore, we demonstrate that both compounds disassemble fibrils extracted from MSA patient brains and prevent their intracellular seeding. They also reduce in vivo alpha-synuclein aggregates in C. elegans . Both compounds also penetrate brain tissue in mice. A molecular dynamics–based computational model suggests the compounds may exert their disaggregating effects on the N terminus of the fibril core. These compounds appear to be promising therapeutic leads for targeting alpha-synuclein for the treatment of synucleinopathies.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2024

Reinforcement expectation in the honeybee ( Apis mellifera ): Can downshifts in reinforcement show conditioned inhibition?

When animals learn the association of a conditioned stimulus (CS) with an unconditioned stimulus (US), later presentation of the CS invokes a representation of the US. When the expected US fails to occur, theoretical accounts predict that conditioned inhibition can accrue to any other stimuli that are associated with this change in the US. Empirical work with mammals has confirmed the existence of conditioned inhibition. But the way it is manifested, the conditions that produce it, and determining whether it is the opposite of excitatory conditioning are important considerations. Invertebrates can make valuable contributions to this literature because of the well-established conditioning protocols and access to the central nervous system (CNS) for studying neural underpinnings of behavior. Nevertheless, although conditioned inhibition has been reported, it has yet to be thoroughly investigated in invertebrates. Here, we evaluate the role of the US in producing conditioned inhibition by using proboscis extension response conditioning of the honeybee (Apis mellifera). Specifically, using variations of a “feature-negative” experimental design, we use downshifts in US intensity relative to US intensity used during initial excitatory conditioning to show that an odorant in an odor–odor mixture can become a conditioned inhibitor. We argue that some alternative interpretations to conditioned inhibition are unlikely. However, we show variation across individuals in how strongly they show conditioned inhibition, with some individuals possibly revealing a different means of learning about changes in reinforcement. We discuss how the resolution of these differences is needed to fully understand whether and how conditioned inhibition is manifested in the honeybee, and whether it can be extended to investigate how it is encoded in the CNS. It is also important for extension to other insect models. In particular, work like this will be important as more is revealed of the complexity of the insect brain from connectome projects.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2023

Identification of ancestral gnathostome Gli3 enhancers with activity in mammals

Abnormal expression of the transcriptional regulator and hedgehog (Hh) signaling pathway effector Gli3 is known to trigger congenital disease, most frequently affecting the central nervous system (CNS) and the limbs. Accurate delineation of the genomic cis-regulatory landscape controlling Gli3 transcription during embryonic development is critical for the interpretation of noncoding variants associated with congenital defects. Here, we employed a comparative genomic analysis on fish species with a slow rate of molecular evolution to identify seven previously unknown conserved noncoding elements (CNEs) in Gli3 intronic intervals (CNE15–21). Transgenic assays in zebrafish revealed that most of these elements drive activities in Gli3 expressing tissues, predominantly the fins, CNS, and the heart. Intersection of these CNEs with human disease associated SNPs identified CNE15 as a putative mammalian craniofacial enhancer, with conserved activity in vertebrates and potentially affected by mutation associated with human craniofacial morphology. Finally, comparative functional dissection of an appendage-specific CNE conserved in slowly evolving fish (elephant shark), but not in teleost (CNE14/hs1586) indicates co-option of limb specificity from other tissues prior to the divergence of amniotes and lobe-finned fish. In conclusion, these results uncover a novel subset of intronic Gli3 enhancers that arose in the common ancestor of gnathostomes and whose sequence components were likely gradually modified in other species during the process of evolutionary diversification.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Human cerebrospinal fluid contains diverse lipoprotein subspecies enriched in proteins implicated in central nervous system health

Lipoproteins in cerebrospinal fluid (CSF) of the central nervous system (CNS) resemble plasma high-density lipoproteins (HDLs), which are a compositionally and structurally diverse spectrum of nanoparticles with pleiotropic functionality. Whether CSF lipoproteins (CSF-Lps) exhibit similar heterogeneity is poorly understood because they are present at 100-fold lower concentrations than plasma HDL. To investigate the diversity of CSF-Lps, we developed a sensitive fluorescent technology to characterize lipoprotein subspecies in small volumes of human CSF. We identified 10 distinctly sized populations of CSF-Lps, most of which were larger than plasma HDL. Mass spectrometric analysis identified 303 proteins across the populations, over half of which have not been reported in plasma HDL. Computational analysis revealed that CSF-Lps are enriched in proteins important for wound healing, inflammation, immune response, and both neuron generation and development. Network analysis indicated that different subpopulations of CSF-Lps contain unique combinations of these proteins. Our study demonstrates that CSF-Lp subspecies likely exist that contain compositional signatures related to CNS health.

59 BASIC BIOLOGICAL SCIENCES↗