Astrocyte Lineage Cells Research Articles

Metabolic Rewiring and Altered Glial Differentiation in an iPSC-Derived Astrocyte Model Derived from a Nonketotic Hyperglycinemia Patient.

The pathophysiology of nonketotic hyperglycinemia (NKH), a rare neuro-metabolic disorder associated with severe brain malformations and life-threatening neurological manifestations, remains incompletely understood. Therefore, a valid human neural model is essential. We aimed to investigate the impact of GLDC gene variants, which cause NKH, on cellular fitness during the differentiation process of human induced pluripotent stem cells (iPSCs) into iPSC-derived astrocytes and to identify sustainable mechanisms capable of overcoming GLDC deficiency. We developed the GLDC27-FiPS4F-1 line and performed metabolomic, mRNA abundance, and protein analyses. This study showed that although GLDC27-FiPS4F-1 maintained the parental genetic profile, it underwent a metabolic switch to an altered serine-glycine-one-carbon metabolism with a coordinated cell growth and cell cycle proliferation response. We then differentiated the iPSCs into neural progenitor cells (NPCs) and astrocyte-lineage cells. Our analysis showed that GLDC-deficient NPCs had shifted towards a more heterogeneous astrocyte lineage with increased expression of the radial glial markers GFAP and GLAST and the neuronal markers MAP2 and NeuN. In addition, we detected changes in other genes related to serine and glycine metabolism and transport, all consistent with the need to maintain glycine at physiological levels. These findings improve our understanding of the pathology of nonketotic hyperglycinemia and offer new perspectives for therapeutic options.

The identification of a Distinct Astrocyte Subtype that Diminishes in Alzheimer's Disease.

Alzheimer's disease (AD) is characterized by the presence of two hallmark pathologies: the accumulation of Amyloid beta (Aβ) and tau proteins in the brain. There is a growing body of evidence suggesting that astrocytes, a type of glial cell in the brain, play crucial roles in clearing Aβ and binding to tau proteins. However, due to the heterogeneity of astrocytes, the specific roles of different astrocyte subpopulations in response to Aβ and tau remain unclear. To enhance the understanding of astrocyte subpopulations in AD, we investigated astrocyte lineage cells based on single-nuclei transcriptomic data obtained from both human and mouse samples. We characterized the diversity of astrocytes and identified global and subpopulation-specific transcriptomic changes between control and AD samples. Our findings revealed the existence of a specific astrocyte subpopulation marked by low levels of GFAP and the presence of AQP4 and CD63 expression, which showed functional enrichment in Aβ clearance and tau protein binding, and diminished in AD. We verified this type of astrocytes in mouse models and in AD patient brain samples. Furthermore, our research also unveiled significant alterations of the ligand-receptor interactions between astrocytes and other cell types. These changes underscore the complex interplay between astrocytes and neighboring cells in the context of AD. Overall, our work gives insights into astrocyte heterogeneity in the context of AD and reveals a distinct astrocyte subpopulation that holds potential for therapeutic interventions in AD. Targeting specific astrocyte subpopulations may offer new avenues for the development of novel treatments for AD.

BMPRII+ neural precursor cells isolated and characterized from organotypic neurospheres: an in vitro model of human fetal spinal cord development.

Roof plate secretion of bone morphogenetic proteins (BMPs) directs the cellular fate of sensory neurons during spinal cord development, including the formation of the ascending sensory columns, though their biology is not well understood. Type-II BMP receptor (BMPRII), the cognate receptor, is expressed by neural precursor cells during embryogenesis; however, an in vitro method of enriching BMPRII+ human neural precursor cells (hNPCs) from the fetal spinal cord is absent. Immunofluorescence was undertaken on intact second-trimester human fetal spinal cord using antibodies to BMPRII and leukemia inhibitory factor (LIF). Regions of highest BMPRII+ immunofluorescence localized to sensory columns. Parenchymal and meningeal-associated BMPRII+ vascular cells were identified in both intact fetal spinal cord and cortex by co-positivity with vascular lineage markers, CD34/CD39. LIF immunostaining identified a population of somas concentrated in dorsal and ventral horn interneurons, mirroring the expression of LIF receptor/CD118. A combination of LIF supplementation and high-density culture maintained culture growth beyond 10 passages, while synergistically increasing the proportion of neurospheres with a stratified, cytoarchitecture. These neurospheres were characterized by BMPRII+/MAP2ab+/-/βIII-tubulin+/nestin-/vimentin-/GFAP-/NeuN- surface hNPCs surrounding a heterogeneous core of βIII-tubulin+/nestin+/vimentin+/GFAP+/MAP2ab-/NeuN- multipotent precursors. Dissociated cultures from tripotential neurospheres contained neuronal (βIII-tubulin+), astrocytic (GFAP+), and oligodendrocytic (O4+) lineage cells. Fluorescence-activated cell sorting-sorted BMPRII+ hNPCs were MAP2ab+/-/βIII-tubulin+/GFAP-/O4- in culture. This is the first isolation of BMPRII+ hNPCs identified and characterized in human fetal spinal cords. Our data show that LIF combines synergistically with high-density reaggregate cultures to support the organotypic reorganization of neurospheres, characterized by surface BMPRII+ hNPCs. Our study has provided a new methodology for an in vitro model capable of amplifying human fetal spinal cord cell numbers for > 10 passages. Investigations of the role BMPRII plays in spinal cord development have primarily relied upon mouse and rat models, with interpolations to human development being derived through inference. Because of significant species differences between murine biology and human, including anatomical dissimilarities in central nervous system (CNS) structure, the findings made in murine models cannot be presumed to apply to human spinal cord development. For these reasons, our human in vitro model offers a novel tool to better understand neurodevelopmental pathways, including BMP signaling, as well as spinal cord injury research and testing drug therapies.

Transplanted neural lineage cells derived from dental pulp stem cells promote peripheral nerve regeneration.

Cell therapy for peripheral nerve injury is a promising strategy as regenerative medicine that restores neurological function. However, challenges remain in producing suitable and sufficient amounts of autologous cells for promoting nerve regeneration. This study aimed to identify the characteristics of neural lineage cells (NLCs) differentiated from dental pulp stem cells (DPSCs) and reveal their effect on functional recovery and nerve regeneration after cell transplantation into an immunodeficient rat using a nerve guide conduit. Here we report a protocol of neural induction in monolayer culture and characterize NLCs in vitro. Furthermore, NLCs were transplanted into an immunodeficient rat model with a 10-mm sciatic nerve defect, and cell survival and differentiation were investigated in vivo. Outcomes of nerve regeneration were also assessed using the remyelinated axon numbers, myelin sheath thickness, electrophysiological activities, and gastrocnemius muscle mass. NLCs comprised neuronal, astrocyte, oligodendrocyte, and neural crest lineage cells. NLCs enhanced the activities of endothelial cells, Schwann cells, and neurons in a paracrine-dependent manner in vitro. At 2weeks post-transplantation, numerous transplanted NLCs differentiated into platelet-derived growth factor receptor alpha (PDGFRα) + oligodendrocyte progenitor cells (OPCs) and a few PDGFRα + /p75 neurotrophin receptor + Schwann cell-like cells derived from OPCs were observed. At 12weeks post-transplantation, human Schwann cell-like cells survived, and axon growth, remyelination, electrophysiological activities, and muscle atrophy were improved. This study demonstrates the broad application of our protocol of neural induction of DPSCs and portrays the efficacy of transplantation of NLCs derived from human DPSCs as a promising strategy for peripheral nerve regeneration.

Abstract WP148: The Effect of Mitochondrial Migration From Astrocyte to Glial Cells Under Prolonged Cerebral Hypoperfusion

Introduction: Astrocytes play broad roles in the Central nervous system, and are involved in the regulation of cerebral metabolism and blood flow. Normal astrocytes (A2) protect against oxidative stress and excitotoxicity, but unhealthy astrocytes (A1) may release deleterious factors. Oligodendrocytes (OLGs) differentiate from oligodendrocyte-precursor-cells (OPCs) for myelination in white matter, but OPC were vulnerable for ischemia. Therefore, differentiation is impaired when white matter injury occurs in a chronic cerebral hypoperfusion model. Thus, we examined the effects of the interaction between astrocyte and oligodendrocyte lineage cells on myelination focused on mitochondrial migration. Method: A microcoil was applied to the bilateral common carotid arteries in male C57BL/6 mice as an in vivo cerebral chronic hypoperfusion model (BCAS model). A nonlethal concentration of CoCl 2 was added to the primary cell culture from the postnatal rat cortex and incubated in vitro. Results: White matter injury progressed in the BCAS model as myelin decreased. The numbers of OPCs and astrocytes increased after the operation, whereas that of OLGs decreased at day 28. Increased astrocytes were mainly A1 type, and A2 type were decreased. OPC differentiation was disrupted under the stressed conditions in the cell culture, but improved after administration of astrocyte-conditioned medium (ACM), but injured ACM couldn’t improve maturation. Incubate with CoCl 2 change astrocyte A2 to A1, and mitochondrial migration also reduced. Trkβ agonist could change astrocyte A1 to A2 even in hyperperfused condition, and also help OPC maturation via mitochondrial migration and drug effect in vivo and in vitro. Conclusions: The reduction in incrementing A1 astrocytes protect white matter injury. and Trkβ agonist may play an important role in the impairment under chronic ischemic conditions. Mitochondrial migration could be a broad therapeutic strategy for cerebrovascular disorders.

Astrocyte lineage cells are essential for functional neuronal differentiation and synapse maturation in human iPSC-derived neural networks.

Human astrocytes differ dramatically in cell morphology and gene expression from murine astrocytes. The latter are well known to be of major importance in the formation of neuronal networks by promoting synapse maturation. However, whether human astrocyte lineage cells have a similar role in network formation has not been firmly established. Here, we investigated the impact of human astrocyte lineage cells on the functional maturation of neural networks that were derived from human induced pluripotent stem cells (hiPSCs). Initial in vitro differentiation of hiPSC-derived neural progenitor cells and immature neurons (glia+ cultures) resulted in spontaneously active neural networks as indicated by synchronous neuronal Ca2+ transients. Depleting proliferating neural progenitors from these cultures by short-term antimitotic treatment resulted in strongly astrocyte lineage cell-depleted neuronal networks (glia- cultures). Strikingly, in contrast to glia+ cultures, glia- cultures did not exhibit spontaneous network activity. Detailed analysis of the morphological and electrophysiological properties of neurons by patch clamp recordings revealed reduced dendritic arborization in glia- cultures. In addition, a reduced action potential frequency upon current injection in pyramidal-like neurons was observed, whereas the electrical excitability of multipolar neurons was unaltered. Furthermore, we found a reduced dendritic density of PSD95-positive excitatory synapses, and more immature properties of AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) miniature excitatory postsynaptic currents (mEPSCs) in glia- cultures, suggesting that the maturation of glutamatergic synapses depends on the presence of hiPSC-derived astrocyte lineage cells. Intriguingly, addition of the astrocyte-derived synapse maturation inducer cholesterol increased the dendritic density of PSD95-positive excitatory synapses in glia- cultures.

The Effects of Astrocyte and Oligodendrocyte Lineage Cell Interaction on White Matter Injury under Chronic Cerebral Hypoperfusion

Oligodendrocytes (OLGs) differentiate from oligodendrocyte-precursor-cells (OPCs) for myelination in white matter. This differentiation is maintained by cell–cell interactions through trophic factors such as brain-derived-neurotrophic-factor (BDNF). However, differentiation is impaired when white matter injury occurs in a chronic cerebral hypoperfusion model. Thus, we examined the effects of the interaction between astrocyte and oligodendrocyte lineage cells on myelination regarding the mechanism of impairment. A microcoil was applied to the bilateral common carotid arteries in male C57BL/6 mice as an in vivo cerebral chronic hypoperfusion model (BCAS model). A nonlethal concentration of CoCl2 was added to the primary cell culture from the postnatal rat cortex and incubated in vitro. White matter injury progressed in the BCAS model as myelin decreased. The numbers of OPCs and astrocytes increased after the operation, whereas that of OLGs decreased at day 28. BDNF continuously decreased until day 28. Differentiation was disrupted under the stressed conditions in the cell culture, but improved after administration of astrocyte-conditioned medium containing BDNF. Astrocytes with BDNF underwent differentiation, but differentiation was impaired under the stressed conditions due to the reduction of BDNF. We examined S100B regarding the mechanism of impairment. S100B is mainly expressed by mature astrocytes, and has neuroprotective and neurotoxic effects inside and outside of cells. GFAP-positive astrocytes increased in the corpus callosum in the BCAS model, whereas the number of mature astrocytes continued to decrease, resulting in reduced BDNF. The reduction in mature astrocytes due to the discharge of S100B in ischemic conditions caused the reduction in BDNF.

Adult neural stem cell isolation from whole rat brain and neurosphere culture and differentiation.

Historically, it was thought that neurogenesis ceased by the end of development, but it is currently understood that neurogenesis continues throughout the life of an individual. This continued growth arises from neural stem or progenitor cells (NSCs) located in specific regions of the adult brain, including the subventricular zone and the dentate gyrus of the hippocampus. Increased understanding of the nature of these cells and their reaction to environmental stimuli is of paramount importance in the effort to discern their role and potential use in repair following neurological disruption. Neurosphere suspension culture is identified as an effective way of actualizing a self-renewing population of neural stem cells. This study demonstrated that adult rat neural stem cells could be effectively induced to differentiate into cells of astrocytic lineage through exposure to fetal bovine serum (FBS), and that the same population of precursor cells could be induced toward neuronal lineage through exposure to dibutyryl cyclic adenosine monophosphate (dcAMP). There were also observations noted regarding difficulty inducing cell attachment following enzymatic digestion of neurospheres, and potential effects on various types of assays, including migration assays (Fig. 7, Ref. 31). Keywords: neural, stem cell, neurosphere, adult rat, cell culture, cell differentiation.

Rare but Recurrent ROS1 Fusions Resulting From Chromosome 6q22 Microdeletions are Targetable Oncogenes in Glioma.

Gliomas, a genetically heterogeneous group of primary central nervous system tumors, continue to pose a significant clinical challenge. Discovery of chromosomal rearrangements involving kinase genes has enabled precision therapy, and improved outcomes in several malignancies. Positing that similar benefit could be accomplished for patients with brain cancer, we evaluated The Cancer Genome Atlas (TCGA) glioblastoma dataset. Functional validation of the oncogenic potential and inhibitory sensitivity of discovered ROS1 fusions was performed using three independent cell-based model systems, and an in vivo murine xenograft study. In silico analysis revealed previously unreported intrachromosomal 6q22 microdeletions that generate ROS1-fusions from TCGA glioblastoma dataset. ROS1 fusions in primary glioma and ependymoma were independently corroborated from MSK-IMPACT and Foundation Medicine clinical datasets. GOPC-ROS1 is a recurrent ROS1 fusion in primary central nervous system (CNS) tumors. CEP85L-ROS1 and GOPC-ROS1 are transforming oncogenes in cells of astrocytic lineage, and amenable to pharmacologic inhibition with several ROS1 inhibitors even when occurring concurrently with other cancer hotspot aberrations frequently associated with glioblastoma. Oral monotherapy with a brain-permeable ROS1 inhibitor, lorlatinib, significantly prolonged survival in an intracranially xenografted tumor model generated from a ROS1 fusion-positive glioblastoma cell line. Our findings highlight that CNS tumors should be specifically interrogated for these rare intrachromosomal 6q22 microdeletion events that generate actionable ROS1 fusions. ROS1 fusions in primary brain cancer may be amenable for clinical intervention with kinase inhibitors, and this holds the potential of novel treatment paradigms in these treatment-refractory cancer types, particularly in glioblastoma.

Human Astrocyte Maturation Captured in 3D Cerebral Cortical Spheroids Derived from Pluripotent Stem Cells

There is significant need to develop physiologicallyrelevant models for investigating human astrocytes in health and disease. Here, we present an approach for generating astrocyte lineage cells in a three-dimensional (3D) cytoarchitecture using human cerebral cortical spheroids (hCSs) derived from pluripotent stem cells. We acutely purified astrocyte-lineage cells from hCSs at varying stages up to 20months invitro using immunopanning and cell sorting and performed high-depth bulk and single-cell RNA sequencing to directly compare them to purified primary human brain cells. We found that hCS-derived glia closely resemble primary human fetal astrocytes and that, over time invitro, they transition from a predominantly fetal to an increasingly mature astrocyte state. Transcriptional changes in astrocytes are accompanied by alterations in phagocytic capacity and effects on neuronal calcium signaling. These findings suggest that hCS-derived astrocytes closely resemble primary human astrocytes and can be used for studying development and modeling disease.

Comparison of methods for deriving neural progenitor cells from nonhuman primate embryonic stem cells

Background: Feeder-free monolayer culture and the suspension culture of embryoid bodies (EB) followed by adherent culture, rosette selection, and expansion are 2 methods for deriving neural progenitor cells (NPCs). Direct comparison of these 2 methods has not yet been reported. Objectives: To compare the influence of NPC derivation methods on the properties of NPCs derived from rhesus monkey (Macaca mulatta) embryonic stem cells (rhESCs). Methods: rhESCs were used to derive NPC lines using 2 different methods. EB were produced from a suspension culture of rhESC clumps and rhESCs were cultured in feeder-free monolayers on poly-L-ornithine/laminin coated plates. NPCs were derived by exposure to induction factors. Cell morphology, neural and nonneural lineage markers were evaluated. We measured the expression of nuclear receptor tailless (TLX), which acts as repressor of glial fibrillary acidic protein (GFAP) expression. Results: NPCs were successful derived using either method, with homogenous populations based on the expression of nestin (>97%) and Pax6 (>99%) as shown by flow cytometry. No significant difference in NPC specific markers or ability to differentiate into neurons in vitro was found between the methods. However, the expression of GFAP was >400-fold higher in cells produced by the feeder-free method. This distinction was consistent with the lower expression of TLX. Conclusions: NPCs derived by feeder-free and EB methods share similar morphology and properties. The elevated expression of GFAP and reduced expression of TLX in NPCs derived using the feeder-free method may explain their greater heterogeneity and tendency to differentiate toward cells of an astrocyte lineage. Keywords: Embryonic stem cells, neural progenitor cells, neural progenitor cell derivation, nonhuman primate,TLX

Abstract A18: Changes in cholesterol metabolism support high-density cell growth and therapeutic resistance in glioblastoma multiforme

Abstract Glioblastoma Multiforme (GBM) is a highly aggressive, therapeutically resistant brain tumor that arises from cells of astrocytic lineage. The five year survival rate is just below 10% and this statistic has changed very little in the past decade. Amplification and activating mutations of receptor tyrosine kinases (RTKs) are common in GBM; however despite their oncogenic importance, RTK inhibition with tyrosine kinase inhibitors (TKIs) has been clinically ineffective for GBM. The gap between promising laboratory results and clinical success is a common obstacle in cancer research in part because cell culture models do not accurately mimic complex in vivo conditions and cell behavior, such as therapeutic resistance. Many cell culture studies and high throughput screens of pharmacological agents are performed in cells plated at sub-confluence, or low density. We have found that GBM tumor initiating cells (GBM-TICs) are sensitive to TKIs at low density; however as culture density increases they become resistant, despite equal inhibition of the target RTK and downstream signals. Thus, culturing cells at low and high density provides a novel, isogenic system for studying the cellular mechanisms of therapeutic response. While density does not affect the cell cycle profile of GBM-TICs, we have found that high density cells have lower levels of reactive oxygen species and ATP than low density cells and reduced mitochondrial membrane potential. This suggests a switch to aerobic glycolysis (the Warburg Effect); however lactate levels, which should increase under conditions of aerobic glycolysis, are unaffected by cell density. Additionally, assessment of extracellular metabolic flux does not support an increase in aerobic glycolysis in dense GBM-TICs. Extracellular acidification rate (ECAR), which should increase with aerobic glycolysis, does not show a tumor-specific effect. Oxygen consumption rate (OCR), which should decrease during aerobic glycolysis, increases significantly with density in GBM-TICs but shows no density-dependent change in healthy astrocytes. These data indicate a tumor-specific, non-Warburg metabolic shift in dense GBM-TICs that is not present in sparse GBM-TICs or normal human astrocytes at any density. Further analysis has implicated changes in cholesterol metabolism and transport in density-dependent metabolic change. Specifically, densely cultured GBM-TICs have higher levels of cholesterol and elevated expression of cholesterol transport proteins such as ABCA1 and ABCG1 than cells cultured sparsely. Pharmacologic inhibition of cholesterol biosynthesis results in reduced OCR and increased ATP/cell in dense but not sparse GBM-TICs, which corroborates the importance of cholesterol synthesis and metabolism in dense GBM-TICs. We are currently investigating the connection between cholesterol metabolism and therapeutic resistance in GBM in the hope of identifying novel therapeutic targets to improve clinical outcomes for GBM patients. Citation Format: Diane M. Kambach, Tamalee Kramp, Jie Liu, Michael L. Oshinsky, Federico Bernal, Jayne M. Stommel. Changes in cholesterol metabolism support high-density cell growth and therapeutic resistance in glioblastoma multiforme. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A18.

S100A6 (calcyclin) is a novel marker of neural stem cells and astrocyte precursors in the subgranular zone of the adult mouse hippocampus

S100A6 (calcyclin), an EF-hand calcium binding protein, is considered to play various roles in the brain, for example, cell proliferation and differentiation, calcium homeostasis, and neuronal degeneration. In addition to some limbic nuclei, S100A6 is distributed in the rostral migratory stream, one of the major neurogenic niches of the adult brain. However, the potential involvement of S100A6 in adult neurogenesis remains unclear. In this study, we aimed to elucidate the role of S100A6 in the other major neurogenic niche, the subgranular zone of the dentate gyrus in the adult mouse hippocampus. Immunofluorescent multiple labeling showed that S100A6 was highly expressed in neural stem cells labeled by sex determining region Y-box 2, brain lipid-binding protein protein and glial fibrillary acidic protein. S100A6+ cells often extended a long process typical of radial glial morphology. In addition, S100A6 was found in some S100β+ astrocyte lineage cells. Interestingly, proliferating cell nuclear antigen was detected in a fraction of S100A6+/S100β+ cells. These cells were considered to be lineage-restricted astrocyte precursors maintaining mitotic potential. On the other hand, S100A6 was rarely seen in neural lineage cells labeled by T-box brain protein 2, doublecortin, calretinin and calbindin D28K. Cell fate-tracing experiment using BrdU showed that the majority of newly generated immature astrocytes were immunoreactive for S100A6, while mature astrocytes lacked S100A6 immunoreactivity. Administration of S100 protein inhibitor, trifluoperazine, caused a reduction in production of S100β+ astrocyte lineage cells, but had no impact on neurogenesis. Overall, our data provide the first evidence that S100A6 is a specific marker of neural stem cells and astrocyte precursors, and may be especially important for generation of astrocytes in the adult hippocampus.

Prion Replication Elicits Cytopathic Changes in Differentiated Neurosphere Cultures

The molecular mechanisms of prion-induced cytotoxicity remain largely obscure. Currently, only a few cell culture models have exhibited the cytopathic changes associated with prion infection. In this study, we introduced a cell culture model based on differentiated neurosphere cultures isolated from the brains of neonatal prion protein (PrP)-null mice and transgenic mice expressing murine PrP (dNP0 and dNP20 cultures). Upon exposure to mouse Chandler prions, dNP20 cultures supported the de novo formation of abnormal PrP and the resulting infectivity, as assessed by bioassays. Furthermore, this culture was susceptible to various prion strains, including mouse-adapted scrapie, bovine spongiform encephalopathy, and Gerstmann-Sträussler-Scheinker syndrome prions. Importantly, a subset of the cells in the infected culture that was mainly composed of astrocyte lineage cells consistently displayed late-occurring, progressive signs of cytotoxicity as evidenced by morphological alterations, decreased cell viability, and increased lactate dehydrogenase release. These signs of cytotoxicity were not observed in infected dNP0 cultures, suggesting the requirement of endogenous PrP expression for prion-induced cytotoxicity. Degenerated cells positive for glial fibrillary acidic protein accumulated abnormal PrP and exhibited features of apoptotic death as assessed by active caspase-3 and terminal deoxynucleotidyltransferase nick-end staining. Furthermore, caspase inhibition provided partial protection from prion-mediated cell death. These results suggest that differentiated neurosphere cultures can provide an in vitro bioassay for mouse prions and permit the study of the molecular basis for prion-induced cytotoxicity at the cellular level.

The astrocytic lineage marker calmodulin‐regulated spectrin‐associated protein 1 (Camsap1): Phenotypic heterogeneity of newly born Camsap1‐expressing cells in injured mouse brain

Calmodulin-regulated spectrin-associated protein 1 (Camsap1) has been recognized as a new marker for astrocytic lineage cells and is expressed on mature astrocytes in the adult brain (Yamamoto et al. [2009] J. Neurosci. Res. 87:503–513). In the present study, we found that newly born Camsap1-expressing cells exhibited regional heterogeneity in an early phase after stab injury of the mouse brain. In the surrounding area of the lesion site, Camsap1 was expressed on quiescent astrocytes. At 3 days after injury, Camsap1 immunoreactivity was upregulated on glial fibrillary acidic protein-immunoreactive (GFAP-ir) astrocytes. Some of these astrocytes incorporated bromodeoxyuridine (BrdU) together with re-expression of the embryonic cytoskeleton protein nestin. In the neighboring region of the lesion cavity, Camsap1 was expressed on GFAP-negative cells. At 3 days after injury, GFAP-ir astrocytes were absent around the lesion cavity. At this stage, NG2-ir cells immunopositive for Camsap1 and immunonegative for GFAP were distributed in border of the lesion cavity. By 10 days, Camsap1 immunoreactivity was exclusively detected on GFAP-ir reactive astrocytes devoid of NG2 immunoreactivity. BrdU pulse-chase labeling assay suggested the differentiation of Camsap1+/NG2+ cells into Camsap1+/GFAP+ astrocytes. In the subependymal zone of the lateral ventricle, Camsap1-ir cells increased after injury. Camsap1 immunoreactivity was distributed on ependymal and subependymal cells bearing various astrocyte markers, and BrdU incorporation was enhanced on such Camsap1-ir cells after injury. These results suggest that newly born reactive astrocytes are derived from heterogeneous Camsap1-expressing cells in the injured brain.

PepT2 transporter protein expression in human neoplastic glial cells and mediation of fluorescently tagged dipeptide derivative β-Ala-Lys-Nε-7-amino-4-methyl-coumarin-3-acetic acid accumulation

The present study was aimed at analyzing the accumulation of the fluorescently tagged dipeptide derivative, beta-Ala-Lys-N(epsilon)-7-amino-4-methyl coumarin-3-acetic acid (AMCA), in primary cultures of human neoplastic glial cells. This molecule is a highly specific reporter used to investigate the dipeptide transport system hPepT2. In this study the authors used immunocytochemical methods to determine the cell-specific accumulation of a small and fluorescently tagged reporter molecule named beta-Ala-Lys-N(epsilon)-AMCA to detect dipeptide transport capacity of neoplastic glial cells. Furthermore, specific mRNA levels were quantified using Northern blot analysis and the tissue distribution of hPepT2 mRNA transcripts was demonstrated with in-situ hybridization histochemical analysis. Recent fluorescent immunocytochemical analyses have revealed that beta-Ala-Lys-N(epsilon)-AMCA specifically accumulates within anaplastic cells of astrocytic lineage but not in anaplastic oligodendrocytes or neurons. Northern blot analysis demonstrated that human hPepT2 mRNA is specifically detected in primary cell cultures of human glioblastoma but not in oligodendroglioma. Moreover, in situ hybridization analyses revealed an astrocytic localization of hPepT2 transcripts in human glioblastoma and astrocytoma cells. The hPepT2 transcription levels were clearly dependent on the grade of glial cell differentiation: within low-grade gliomas (WHO Grade II), more hPepT2 mRNA was detected compared with tumors of a higher grade of dedifferentiation (WHO Grade IV). Analysis of expression levels of hPepT2 mRNA in human neoplastic glial cells xenografted into the brains of athymic rats (han rnu(+/+)) showed a markedly increased expression of hPepT2 after 2 weeks of growth in vivo compared with the primary counterparts grown in vitro. The authors concluded that expression of the hPepT2 transporter protein is a characteristic of glial cells of astrocytic lineage, and is dependent on the grade of astroglial cell differentiation and the extracellular matrix (here brain neuropil). The authors found that beta-Ala-Lys-N(epsilon)-AMCA is as an excellent reporter molecule for assessing neoplastic glial cell function and physiological characteristics.

Olig2-expressing progenitor cells preferentially differentiate into oligodendrocytes in cuprizone-induced demyelinated lesions

Many oligodendrocyte progenitor cells (OPCs) are found in acute or chronic demyelinated area, but not all of them differentiate efficiently into mature oligodendrocytes in the demyelinated central nervous system (CNS). Recent studies have shown that the basic helix-loop-helix transcription factor Olig2, which stimulates OPCs to differentiate into oligodendrocyte, is strongly up-regulated in many pathological conditions including acute or chronic demyelinating lesions in the adult CNS. Despite their potential role in the treatment of demyelinating diseases, the long-term fate of these up-regulated Olig2 cells has not been identified due to the lack of stable labeling methods. To trace their fate we have used double-transgenic mice, in which we were able to label Olig2-positive cells conditionally with green fluorescent protein (GFP). Demyelination was induced in these mice by feeding cuprizone, a copper chelator. After 6 weeks of cuprizone exposure, GFP-positive (GFP +) cells were processed for a second labeling with antibodies to major neural cell markers APC (mature oligodendrocyte marker), GFAP (astrocyte marker), NeuN (neuron marker), Iba1 (microglia marker) and NG2 proteoglycan (oligodendrocyte progenitor marker). More than half of the GFP + cells in the external capsule showed co-localization with NG2 proteoglycan. While the percentages of NG2-positive (NG2 +) and APC-positive (APC +) oligodendrocyte lineage cells in cuprizone-treated mice were significantly higher than those in the normal diet group, no significant difference was observed for GFAP-positive (GFAP +) astrocytic lineage cells. Our data therefore provide direct evidence that proliferation and differentiation of local and/or recruited Olig2 progenitors contribute to remyelination in demyelinated lesions.

Control of glial precursor cell development in the mouse optic nerve by sonic hedgehog from retinal ganglion cells

The development of glial precursor cells in the mammalian optic nerve depends on retinal ganglion cell (RGC) axons, but the signals that mediate this neuron-to-glia interaction have not been fully characterized. Sonic hedgehog (Shh) is expressed by RGCs, and we showed previously that it is required for the specification of astrocyte lineage cells at the optic disc. To study the role of RGC-derived Shh on astrocyte development at later developmental stages, we generated mice with a conditional ablation of Shh in the peripheral retina and analyzed gene expression and glial cell development in the optic nerve. Astrocyte development was initiated in the optic nerves of these mutant mice; however, the expression of Hedgehog (Hh) target genes, Gli1 and Ptch1 and cell cycle genes, Ccnd1 and Cdc25b in the optic nerves were downregulated. Astrocyte proliferation was markedly reduced. Oligodendrocyte precursor cells were fewer in the optic nerves of mutant mice, possibly as a consequence of reduced secretion of growth factors by astrocytes. At a later developmental stage, optic nerve axons displayed signs of Wallerian degeneration, including reduction of astrocyte processes, degenerating glial cells and formation of distended axons. We also demonstrate that the Hh pathway can be activated in optic nerve-derived astrocytes in vitro, but fails to induce cell cycle gene expression and proliferation. RGC-derived Shh signalling isthus necessary in vivo for maintenance of astrocyte proliferation, affecting both axo-glial and normal glial cell development in the optic nerve.

The inhibitor of DNA binding 2 is mainly expressed in oligodendrocyte lineage cells in adult rat brain

The inhibitor of DNA binding 2 (Id2) plays an important role in the brain both during embryogenesis and adulthood. But in adult rat brain, it is still unknown whether Id2 immunoreactivity mainly exhibits in neuronal, astrocytic and/or oligodendrocyte lineage cells. It is also unclear where and when Id2 immunoreactivity mainly exhibits in oligodendrocyte lineage cells. The present study showed 90% of Id2-immunoreactivity in oligodendrocyte lineage cells in such brain regions as the corpus callosum, optic chiasm, the longitudinal fasciculus of pons, the medial septal nucleus, the fimbria of hippocampus, the anterior commissure, and the pyramidal tract. Five percent of Id2-immunoreactivity was found in astrocytes. Id2 immunoreactivity was localized in neurons of only a few brain regions. Seventy percent of Id2 immunoreactivity was found in CC-1-positive mature oligodendrocytes. These observations suggest that Id2 may be mainly involved in terminal maturation of oligodendrocytes and myelination.

Does Vasculogenic Mimicry Exist in Astrocytoma?

Vasculogenic mimicry (VM) has been observed in melanoma and in some nonmelanoma tumor types. It is unknown whether a similar VM phenomenon exists in astrocytoma. The present study was to examine 45 astrocytomas (including World Health Organization grade II 15 cases, grade III 15 cases, and grade IV 15 cases) by CD34 endothelial marker periodic acid-Schiff (PAS) dual staining to see if VM existing in these tumors. The results demonstrated that endothelium-lined vessels dominated the tumor microvasculature and stained positively for PAS, laminin, and endothelial marker. PAS-positive pattern of VM was found in two grade IV astrocytomas. Channels stained positively for PAS, laminin, and negatively for CD34 of the VM entrapped in the tumor tissue. Erythrocytes could be observed in some of these channels. In these networks of PAS-positive pattern, spots of weak reaction for CD34 were observed, suggesting the incorporation of VM channel and normal vessel. Furthermore, in astrocytoma, especially glioblastoma, focus of anaplastic tumor cells appeared with CD34 expression, whereas some tumor cells lost glial fibrillary acid protein expression. It is assumed that genetically deregulated tumor cells in astrocytoma could lose the astrocyte-specific protein and express inappropriate markers not expected in cells of astrocyte lineage. The present results suggest that VM phenomenon exists in some malignant astrocytoma.