Space Microgravity Alters Neural Stem Cell Division: Implications for Brain Cancer Research on Earth and in Space.

Considering the imminence of long-term space travel, it is necessary to investigate the impact of space microgravity (SPC-µG) in order to determine if this environment has consequences on the astronauts' health, in particular, neural and cognitive functions. Neural stem cells (NSCs) are the basis for the regeneration of the central nervous system (CNS) cell populations and learning how weightlessness impacts NSCs in health and disease provides a critical tool for the potential mitigation of specific mechanisms leading to neurological disorders. In previous studies, we found that exposure to SPC-µG resulted in enhanced proliferation, a shortened cell cycle, and a larger cell diameter of NSCs compared to control cells. Here, we report the frequent occurrence of abnormal cell division (ACD) including incomplete cell division (ICD), where cytokinesis is not successfully completed, and multi-daughter cell division (MDCD) of NSCs following SPC-µG as well as secretome exposure compared to ground control (1G) NSCs. These findings provide new insights into the potential health implications of space travel and have far-reaching implications for understanding the mechanisms leading to the deleterious effects of long-term space travel as well as potential carcinogenic susceptibility. Knowledge of these mechanisms could help to develop preventive or corrective measures for successful long-term SPC-µG exposure.

Simulated microgravity enhances oligodendrocyte mitochondrial function and lipid metabolism.

The primary energy sources of mammalian cells are proteins, fats, and sugars that are processed by well-known biochemical mechanisms that have been discovered and studied in 1G (terrestrial gravity). Here we sought to determine how simulated microgravity (sim-µG) impacts both energy and lipid metabolism in oligodendrocytes (OLs), the myelin-forming cells in the central nervous system. We report increased mitochondrial respiration and increased glycolysis 24 hr after exposure to sim-µG. Moreover, examination of the secretome after 3 days' exposure of OLs to sim-µG increased the Krebs cycle (Krebs and Weitzman, ) flux in sim-µG. The secretome study also revealed a significant increase in the synthesis of fatty acids and complex lipids such as 1,2-dipalmitoyl-GPC (5.67); lysolipids like 1-oleoyl-GPE (4.48) were also increased by microgravity. Although longer-chain lipids were not observed in this study, it is possible that at longer time points OLs would have continued moving forward toward the synthesis of lipids that constitute myelin. For centuries, basic developmental biology research has been the pillar of an array of discoveries that have led to clinical applications; we believe that studies using microgravity will open new avenues to our understanding of the brain in health and disease-in particular, to the discovery of new molecules and mechanisms impossible to unveil while in 1G. © 2016 Wiley Periodicals, Inc.

Proof-of Concept that an Acute Trophic Factors Intervention After Spinal Cord Injury Provides an Adequate Niche for Neuroprotection, Recruitment of Nestin-Expressing Progenitors and Regeneration.

Trophic factor treatment has been shown to improve the recovery of brain and spinal cord injury (SCI). In this study, we examined the effects of TSC1 (a combination of insulin-like growth factor 1 and transferrin) 4 and 8 h after SCI at the thoracic segment level (T12) in nestin-GFP transgenic mice. TSC1 treatment for 4 and 8 h increased the number of nestin-expressing cells around the lesion site and prevented Wallerian degeneration. Treatment with TSC1 for 4 h significantly increased heat shock protein (HSP)-32 and HSP-70 expression 1 and 2 mm from lesion site (both, caudal and rostral). Conversely, the number of HSP-32 positive cells decreased after an 8-h TSC1 treatment, although it was still higher than in both, non-treated SCI and intact spinal cord animals. Furthermore, TSC1 increased NG2 expressing cell numbers and preserved most axons intact, facilitating remyelination and repair. These results support our hypothesis that TSC1 is an effective treatment for cell and tissue neuroprotection after SCI. An early intervention is crucial to prevent secondary damage of the injured SC and, in particular, to prevent Wallerian degeneration.

Space Flight Enhances Stress Pathways in Human Neural Stem Cells.

Mammalian cells have evolved to function under Earth's gravity, but how they respond to microgravity remains largely unknown. Neural stem cells (NSCs) are essential for the maintenance of central nervous system (CNS) functions during development and the regeneration of all CNS cell populations. Here, we examined the behavior of space (SPC)-flown NSCs as they readapted to Earth's gravity. We found that most of these cells survived the space flight and self-renewed. Yet, some showed enhanced stress responses as well as autophagy-like behavior. To ascertain if the secretome from SPC-flown NSCs contained molecules inducing these responses, we incubated naïve, non-starved NSCs in a medium containing SPC-NSC secretome. We found a four-fold increase in stress responses. Proteomic analysis of the secretome revealed that the protein of the highest content produced by SPC-NSCs was secreted protein acidic and rich in cysteine (SPARC), which induces endoplasmic reticulum (ER) stress, resulting in the cell's demise. These results offer novel knowledge on the response of neural cells, particularly NSCs, subjected to space microgravity. Moreover, some secreted proteins have been identified as microgravity sensing, paving a new venue for future research aiming at targeting the SPARC metabolism. Although we did not establish a direct relationship between microgravity-induced stress and SPARC as a potential marker, these results represent the first step in the identification of gravity sensing molecules as targets to be modulated and to design effective countermeasures to mitigate intracranial hypertension in astronauts using structure-based protein design.

Chemotactic and mitogenic stimuli of neuronal apoptosis in patients with medically intractable temporal lobe epilepsy.

To identify the upstream signals of neuronal apoptosis in patients with medically intractable temporal lobe epilepsy (TLE), we evaluated by immunohistochemistry and confocal microscopy brain tissues of 13 TLE patients and 5 control patients regarding expression of chemokines and cell-cycle proteins. The chemokine RANTES (CCR5) and other CC-chemokines and apoptotic markers (caspase-3, -8, -9) were expressed in lateral temporal cortical and hippocampal neurons of TLE patients, but not in neurons of control cases. The chemokine RANTES is usually found in cytoplasmic and extracellular locations. However, in TLE neurons, RANTES was displayed in an unusual location, the neuronal nuclei. In addition, the cell-cycle regulatory transcription factor E2F1 was found in an abnormal location in neuronal cytoplasm. The pro-inflammatory enzyme cyclooxygenase-2 and cytokine interleukin-1ß were expressed both in neurons of patients suffering from temporal lobe epilepsy and from cerebral trauma. The vessels showed fibrin leakage, perivascular macrophages and expression of IL-6 on endothelial cells. In conclusion, the cytoplasmic effects of E2F1 and nuclear effects of RANTES might have novel roles in neuronal apoptosis of TLE neurons and indicate a need to develop new medical and/or surgical neuroprotective strategies against apoptotic signaling by these molecules. Both RANTES and E2F1 signaling are upstream from caspase activation, thus the antagonists of RANTES and/or E2F1 blockade might be neuroprotective for patients with medically intractable temporal lobe epilepsy. The results have implications for the development of new medical and surgical therapies based on inhibition of chemotactic and mitogenic stimuli of neuronal apoptosis in patients with medically intractable temporal lobe epilepsy.

Metabolomic Profiling of the Secretome from Human Neural Stem Cells Flown into Space.

The change in gravitational force has a significant effect on biological tissues and the entire organism. As with any alteration in the environment, microgravity (µG) produces modifications in the system inducing adaptation to the new condition. In this study, we analyzed the effect of µG on neural stem cells (NSCs) following a space flight to the International Space Station (ISS). After 3 days in space, analysis of the metabolome in culture medium revealed increased glycolysis with augmented pyruvate and glycerate levels, and activated catabolism of branched-chain amino acids (BCAA) and glutamine. NSCs flown into space (SPC-NSCs) also showed increased synthesis of NADH and formation of polyamine spermidine when compared to ground controls (GC-NSCs). Overall, the space environment appears to increase energy demands in response to the µG setting.

Metabolomics Profile of the Secretome of Space-Flown Oligodendrocytes.

Intracranial hypertension (ICP) and visual impairment intracranial pressure (VIIP) are some of the sequels of long-term space missions. Here we sought to determine how space microgravity (µG) impacts the metabolomics profile of oligodendrocyte progenitors (OLPs), the myelin-forming cells in the central nervous system. We report increased glutamate and energy metabolism while the OLPs were in space for 26 days. We also show that after space flight, OLPs (SPC OLPs) display significantly increased mitochondrial respiration and glycolysis. These data are in agreement with our previous work using simulated microgravity. In addition, our global metabolomics approach allowed for the discovery of endogenous metabolites secreted by OLPs while in space that are significantly modulated by microgravity. Our results provide, for the first time, relevant information about the energetic state of OLPs while in space and after space flight. The functional and molecular relevance of these specific pathways are promising targets for therapeutic intervention for humans in long-term space missions to the moon, Mars and beyond.

Oligodendrocyte Progenitors Display Enhanced Proliferation and Autophagy after Space Flight.

Intracranial hypertension (ICP) and visual impairment intracranial pressure (VIIP) are some of the consequences of long-term space missions. Here we examined the behavior of oligodendrocyte progenitors (OLPs) after space flight using time-lapse microscopy. We show that most OLPs divided more than ground control (GC) counterparts did. Nonetheless, a subpopulation of OLPs flown to space presented a significant increase in autophagic cell death. Examination of the proteomic profile of the secretome of space flown OLPs (SPC-OLPs) revealed that the stress protein heat shock protein-90 beta "HSP-90ß" was the 5th most enriched (6.8 times) and the secreted protein acidic and rich in cysteine "SPARC" was the 7th most enriched (5.2 times), with respect to ground control cells. SPARC induces endoplasmic reticulum stress, which leads to autophagy. Given the roles and importance of these two proteins in mammalian cells' metabolism, their upregulation may hold the key to modulating cell proliferation and autophagy, in order to mitigate ICP and VIIP during and after space missions.

Correction: Tran et al. Delayed Maturation of Oligodendrocyte Progenitors by Microgravity: Implications for Multiple Sclerosis and Space Flight. Life 2022, 12, 797.

The authors wish to make the following corrections to Figure 10 of this paper [...].

Strategies for endogenous spinal cord repair: HPMA hydrogel to recruit migrating endogenous stem cells.

Injury to the spinal cord disrupts ascending and descending axonal pathways and causes tissue damage with a subsequent limited cellular regeneration. Successful treatment would encompass the restoration of the cytoarchitecture, homeostasis and function all in dear need. Transplantation-based treatments using exogenous cells are the most favoured approach. Yet, with the advent of the stem cell concept and continuous progress in the field it became clear that the endogenous potential for repair is greater than previously thought. As an alternative to neural grafting, we and other researchers have aimed at understanding what are the elements needed for a successful repair with self progenitors that would give rise to the cell types needed to restore function of the central nervous system. Some studies involve both scaffolds and cell grafts. Here we describe studies on spinal cord repair using what we call "endogenous tissue engineering for regenerative medicine". The approach involves a hydrogel that mimics the natural milieu where endogenous pre-existing and newly formed cells populate the gel progressively allowing for the integration of CNS self populations leading to a successful recovery of function. Highlight aspects learned from this type of studies are that: Endogenous reconstruction of the injured spinal cord is possible by using the adequate support. The contribution of nestin-expressing progenitors to spinal cord regeneration is continuous and substantial both, in the reconstructed segment as well as, along the distal and caudal segments of the reconstructed spinal cord. Most of these cells appear to have been in a quiescent state until the injury occurred and only a small fraction of these neural progenitors was produced via cell proliferation. The hydrogel combined with exercise was necessary and sufficient to restore locomotor function in cats that underwent spinal transaction followed by reconstructive surgery. This recovery of function was first seen 28 days after surgery and continued to improve for at least 21 months. Therefore, endogenous pre-existing and newly formed cells populated the gel scaffold established contact with the non injured tissue and lead to recovery of function.

Delayed Maturation of Oligodendrocyte Progenitors by Microgravity: Implications for Multiple Sclerosis and Space Flight.

In previous studies, we examined the effects of space microgravity on human neural stem cells. To date, there are no studies on a different type of cell that is critical for myelination and electrical signals transmission, oligodendrocyte progenitors (OLPs). The purpose of the present study was to examine the behavior of space-flown OLPs (SPC-OLPs) as they were adapting to Earth's gravity. We found that SPC-OLPs survived, and most of them proliferated normally. Nonetheless, some of them displayed incomplete cytokinesis. Both morphological and ontogenetic analyses showed that they remained healthy and expressed the immature OLP markers Sox2, PDGFR-α, and transferrin (Tf) after space flight, which confirmed that SPC-OLPs displayed a more immature phenotype than their ground control (GC) counterparts. In contrast, GC OLPs expressed markers that usually appear later (GPDH, O4, and ferritin), indicating a delay in SPC-OLPs' development. These cells remained immature even after treatment with culture media designed to support oligodendrocyte (OL) maturation. The most remarkable and surprising finding was that the iron carrier glycoprotein Tf, previously described as an early marker for OLPs, was expressed ectopically in the nucleus of all SPC-OLPs. In contrast, their GC counterparts expressed it exclusively in the cytoplasm, as previously described. In addition, analysis of the secretome demonstrated that SPC-OLPs contained 3.5 times more Tf than that of GC cells, indicating that Tf is gravitationally regulated, opening two main fields of study to understand the upregulation of the Tf gene and secretion of the protein that keep OLPs at a progenitor stage rather than moving forward to more mature phenotypes. Alternatively, because Tf is an autocrine and paracrine factor in the central nervous system (CNS), in the absence of neurons, it accumulated in the secretome collected after space flight. We conclude that microgravity is becoming a novel platform to study why in some myelin disorders OLPs are present but do not mature.

Trophic factors are essential for the survival of grafted oligodendrocyte progenitors and for neuroprotection after perinatal excitotoxicity.

The consequences of neonatal white matter injury are devastating and represent a major societal problem as currently there is no cure. Prematurity, low weight birth and maternal pre-natal infection are the most frequent causes of acquired myelin deficiency in the human neonate leading to cerebral palsy and cognitive impairment. In the developing brain, oligodendrocyte (OL) maturation occurs perinatally, and immature OLs are particularly vulnerable. Cell replacement therapy is often considered a viable option to replace progenitors that die due to glutamate excitotoxicity. We previously reported directed specification and mobilization of endogenous committed and uncommitted neural progenitors by the combination of transferrin and insulin growth factor 1 (TSC1). Here, considering cell replacement and integration as therapeutic goals, we examined if OL progenitors (OLPs) grafted into the brain parenchyma of mice that were subjected to an excitotoxic insult could rescue white matter injury. For that purpose, we used a well-established model of glutamate excitotoxic injury. Four-day-old mice received a single intraparenchymal injection of the glutamate receptor agonist N-methyl-D-aspartate alone or in conjunction with TSC1 in the presence or absence of OLPs grafted into the brain parenchyma. Energetics and expression of stress proteins and OL developmental specific markers were examined. A comparison of the proteomic profile per treatment was also ascertained. We found that OLPs did not survive in the excitotoxic environment when grafted alone. In contrast, when combined with TSC1, survival and integration of grafted OLPs was observed. Further, energy metabolism in OLPs was significantly increased by N-methyl-D-aspartate and modulated by TSC1. The proteomic profile after the various treatments showed elevated ubiquitination and stress/heat shock protein 90 in response to N-methyl-D-aspartate. These changes were reversed in the presence of TSC1 and ubiquitination was decreased. The results obtained in this pre-clinical study indicate that the use of a combinatorial intervention including both trophic support and healthy OLPs constitutes a promising approach for long-term survival and successful graft integration. We established optimal conditioning of the host brain environment to promote long-term survival and integration of grafted OLPs into an inflamed neonate host brain. Experimental procedures were performed under the United States Public Health Service Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care Committee at (UCLA) (ARC #1992-034-61) on July 1, 2010.

Alzheimer disease macrophages shuttle amyloid-beta from neurons to vessels, contributing to amyloid angiopathy.

Neuronal accumulation of oligomeric amyloid-beta (Alphabeta) is considered the proximal cause of neuronal demise in Alzheimer disease (AD) patients. Blood-borne macrophages might reduce Abeta stress to neurons by immigration into the brain and phagocytosis of Alphabeta. We tested migration and export across a blood-brain barrier model, and phagocytosis and clearance of Alphabeta by AD and normal subjects' macrophages. Both AD and normal macrophages were inhibited in Alphabeta export across the blood-brain barrier due to adherence of Abeta-engorged macrophages to the endothelial layer. In comparison to normal subjects' macrophages, AD macrophages ingested and cleared less Alphabeta, and underwent apoptosis upon exposure to soluble, protofibrillar, or fibrillar Alphabeta. Confocal microscopy of stained AD brain sections revealed oligomeric Abeta in neurons and apoptotic macrophages, which surrounded and infiltrated congophilic microvessels, and fibrillar Abeta in plaques and microvessel walls. After incubation with AD brain sections, normal subjects' monocytes intruded into neurons and uploaded oligomeric Abeta. In conclusion, in patients with AD, macrophages appear to shuttle Abeta from neurons to vessels where their apoptosis may release fibrillar Abeta, contributing to cerebral amyloid angiopathy.

Human Neural Stem Cells Flown into Space Proliferate and Generate Young Neurons.

Here we demonstrate that human neural stem cells (NSCs) proliferate while in space and they express specific NSC markers after being in space. NSCs displayed both higher oxygen consumption and glycolysis than ground controls. These cells also kept their ability to become young neurons. Electrophysiological recordings of space NSC-derived neurons showed immature cell membrane properties characterized by small capacitance and very high input resistance. Current injections elicited only an incipient action potential. No spontaneous synaptic events could be detected, suggesting their immature status even though most recorded cells displayed complex morphology and numerous cell processes. Ascertaining the origin of the NSCs' increased energy requirement is of the essence in order to design effective measures to minimize health risks associated with long-duration human spaceflight missions.

Calcium receptor expression and function in oligodendrocyte commitment and lineage progression: potential impact on reduced myelin basic protein in CaR-null mice.

Oligodendrocytes develop from oligodendrocyte progenitor cells (OPCs), which in turn arise from a subset of neuroepithelial precursor cells during midneurogenesis. Development of the oligodendrocyte lineage involves a plethora of cell-intrinsic and -extrinsic signals. A cell surface calcium-sensing receptor (CaR) has been shown to be functionally expressed in immature oligodendrocytes. Here, we investigated the expression and function of the CaR during oligodendrocyte development. We show that the order of CaR mRNA expression as assessed by quantitative polymerase chain reaction is mature oligodendrocyte > neuron > astrocyte. We next determined the rank order of CaR expression on inducing specification of neural stem cells to the neuronal, oligodendroglial, or astrocytic lineages and found that the relative levels of CaR mRNA expression are OPC > neuron > astrocytes. CaR mRNA expression in cells at various stages of development along the oligodendrocyte lineage revealed that its expression is robustly up-regulated during the OPC stage and remains high until the premyelinating stage, decreasing thereafter by severalfold in the mature oligodendrocyte. In OPCs, high Ca(2+) acting via the CaR promotes cellular proliferation. We further observed that high Ca(2+) stimulates the mRNA levels of myelin basic protein in preoligodendrocytes, which is also CaR mediated. Finally, myelin basic protein levels were significantly reduced in the cerebellum of CaR-null mice during development. Our results show that CaR expression is up-regulated when neural stem cells are specified to the oligodendrocyte lineage and that activation of the receptor results in OPC expansion and differentiation. We conclude that the CaR may be a novel regulator of oligodendroglial development and function.

Efficient Generation of Viral and Integration-Free Human Induced Pluripotent Stem Cell-Derived Oligodendrocytes.

Here we document three highly reproducible protocols: (1) a culture system for the derivation of human oligodendrocytes (OLs) from human induced pluripotent stem cells (hiPS) and their further maturation-our protocol generates viral- and integration-free OLs that efficiently commit and move forward in the OL lineage, recapitulating all the steps known to occur during in vivo development; (2) a method for the isolation, propagation and maintenance of neural stem cells (NSCs); and (3) a protocol for the production, isolation, and maintenance of OLs from perinatal rodent and human brain-derived NSCs. Our unique culture systems rely on a series of chemically defined media, specifically designed and carefully characterized for each developmental stage of OL as they advance from OL progenitors to mature, myelinating cells. We are confident that these protocols bring our field a step closer to efficient autologous cell replacement therapies and disease modeling. © 2016 by John Wiley & Sons, Inc.

Efficient Generation of Viral and Integration-Free Human Induced Pluripotent Stem Cell-Derived Oligodendrocytes.

Here we document three highly reproducible protocols: (1) a culture system for the derivation of human oligodendrocytes (OLs) from human induced pluripotent stem cells (hiPS) and their further maturation-our protocol generates viral- and integration-free OLs that efficiently commit and move forward in the OL lineage, recapitulating all the steps known to occur during in vivo development; (2) a method for the isolation, propagation and maintenance of neural stem cells (NSCs); and (3) a protocol for the production, isolation, and maintenance of OLs from perinatal rodent and human brain-derived NSCs. Our unique culture systems rely on a series of chemically defined media, specifically designed and carefully characterized for each developmental stage of OL as they advance from OL progenitors to mature, myelinating cells. We are confident that these protocols bring our field a step closer to efficient autologous cell replacement therapies and disease modeling. © 2016 by John Wiley & Sons, Inc.

Stem/progenitor cells in the postnatal inner ear of the GFP-nestin transgenic mouse.

Nestin promoter-GFP (green fluorescent protein) transgenic mice were used to determine the presence of stem/progenitor cells in the mouse inner ear. We examined the inner ear of mice at the following postnatal days (P): P0, P4, P5, P15 and P60. Hair cells stereocilia were identified with the use of the histochemical marker phalloidin. Whole endorgans or cryosections were analyzed under epi-fluorescent or confocal microscopy. From P0 to P5, GFP expressing cells were found in the vestibular sensory epithelia of the macula utricle, but not in the crista ampullaris. Cells within the stroma (tissue underneath the sensory epithelia), utricle, and crista were also GFP-positive. Satellite cells in the vestibular ganglia were GFP-positive, while vestibular ganglia neurons were not. In the organ of Corti, GFP signal was found in inner border and inner phalangeal cells that surround the inner hair cells (GFP-negative), Dieters cells and cells in the great epithelial ridge. Outer hair cells were mildly positive for GFP. Satellite cells in the spiral ganglia were GFP-positive, while spiral ganglia neurons were not. Similar GFP expression was found in the vestibule and cochlea of animals at P15, however, outer hair cells showed no GFP expression. The inner ear of P60 animals contained moderate GFP expression in the stroma of the crista ampullaris and utricle, but not within the sensory epithelia. In the organ of Corti, moderate GFP expression was found in a few Deiters cells. The present data indicates that the expression of nestin in the mouse inner ear is developmentally regulated; yet in the adult inner ear there are some nestin expressing cells, suggesting an intrinsic repair potential, although to a more limited extent than during early post-natal life.

Tumor necrosis factor modulates transcription of myelin basic protein gene through nuclear factor kappa B in a human oligodendroglioma cell line.

Tumor necrosis factor-alpha (TNF-alpha) is a major mediator of inflammation and it is involved in many neurological disorders such as multiple sclerosis. Levels of TNF-alpha and lymphotoxin-alpha have been found elevated in plaques, bloods, and cerebral spinal fluids from multiple sclerosis patients. The expression of myelin basic protein (MBP), a major protein of the myelin sheath, is affected by cytokines secreted by activated immune cells. To determine the signal transduction pathway involving tumor necrosis factor's action in myelination and demyelination, we have cloned and analyzed cis-elements on promoters of the human and mouse MBP genes. There are two putative nuclear factors kappa-B (NF-kappaB) cis-elements on the human and one on the mouse gene promoter. In an electrophoretic mobility shift assay, all three NF-kappaB cis-elements showed binding to a protein, which was recognized by an antibody against NF-kappaB P65 component. The specificity of the binding was demonstrated in a competitive assay using NF-kappaB consensus oligonucleotides. A two base pair site-directed mutation on the mouse NF-kappaB cis-element abolished its binding activity. We created a DNA construct by linking the mouse MBP gene promoter containing the NF-kappaB cis-element to luciferase gene. Transfection of this construct into a human oligodendroglioma cell line showed TNF-alpha increased the transgene expression. Furthermore the mutation of NF-kappaB site abolished TNF-alpha -induction of the transgene. The data demonstrate that NF-kappaB is the mediator between tumor necrosis factor's action and MBP gene expression. Elucidating the molecular mechanisms underlying TNF-alpha regulation of MBP gene expression provides new scientific bases for the development of therapy against oligodendrocyte-specific and myelin-related disorders such as multiple sclerosis.