Astrocyte Precursors Research Articles

MEK Is a Key Regulator of Gliogenesis in the Developing Brain

We have defined functions of MEK in regulating gliogenesis in developing cerebral cortex using loss- and gain-of-function mouse genetics. Radial progenitors deficient in both Mek1 and Mek2 fail to transition to the gliogenic mode in late embryogenesis, and astrocyte and oligodendroglial precursors fail to appear. In exploring mechanisms, we found that the key cytokine-regulated gliogenic pathway is attenuated. Further, the Ets transcription family member Etv5/Erm is strongly regulated by MEK and Erm overexpression can rescue the gliogenic potential of Mek-deleted progenitors. Remarkably, Mek1/2-deleted mice surviving postnatally exhibit cortices almost devoid of astrocytes and oligodendroglia and exhibit neurodegeneration. Conversely, expression of constitutively active MEK1 leads to a major increase in numbers of astrocytes in the adult brain. We conclude that MEK is essential for acquisition of gliogenic competence by radial progenitors and that levels of MEK activity regulate gliogenesis in the developing cortex.

Platelet-Derived Growth Factor Over-Expression in Retinal Progenitors Results in Abnormal Retinal Vessel Formation

Platelet-derived growth factor (PDGF) plays an important role in development of the central nervous system, including the retina. Excessive PDGF signaling is associated with proliferative retinal disorders. We reported previously that transgenic mice in which PDGF-B was over-expressed under control of the nestin enhancer, nes/tk-PdgfB-lacZ, exhibited enhanced apoptosis in the developing corpus striatum. These animals display enlarged lateral ventricles after birth as well as behavioral aberrations as adults. Here, we report that in contrast to the relatively mild central nervous system phenotype, development of the retina is severely disturbed in nes/tk-PdgfB-lacZ mice. In transgenic retinas all nuclear layers were disorganized and photoreceptor segments failed to develop properly. Since astrocyte precursor cells did not populate the retina, retinal vascular progenitors could not form a network of vessels. With time, randomly distributed vessels resembling capillaries formed, but there were no large trunk vessels and the intraocular pressure was reduced. In addition, we observed a delayed regression of the hyaloid vasculature. The prolonged presence of this structure may contribute to the other abnormalities observed in the retina, including the defective lamination.

Regulated temporal-spatial astrocyte precursor cell proliferation involves BRAF signalling in mammalian spinal cord.

Expansion of astrocyte populations in the central nervous system is characteristic of evolutionarily more complex organisms. However, regulation of mammalian astrocyte precursor proliferation during development remains poorly understood. Here, we used Aldh1L1-GFP to identify two morphologically distinct types of proliferative astrocyte precursors: radial glia (RG) in the ventricular zone and a second cell type we call an ‘intermediate astrocyte precursor’ (IAP) located in the mantle region of the spinal cord. Astrogenic RG and IAP cells proliferated in a progressive ventral-to-dorsal fashion in a tight window from embryonic day 13.5 until postnatal day 3, which correlated precisely with the pattern of active ERK signalling. Conditional loss of BRAF function using BLBP-cre resulted in a 20% decrease in astrocyte production, whereas expression of activated BRAFV600E resulted in astrocyte hyperproliferation. Interestingly, BRAFV600E mitogenic effects in astrocytes were restricted, in part, by the function of p16INK4A-p19ARF, which limited the temporal epoch for proliferation. Together, these findings suggest that astrocyte precursor proliferation involves distinct RG and IAP cells; is subjected to temporal and spatial control; and depends in part on BRAF signalling at early stages of mammalian spinal cord development.

Regulated temporal-spatial astrocyte precursor cell proliferation involves BRAF signalling in mammalian spinal cord

Expansion of astrocyte populations in the central nervous system is characteristic of evolutionarily more complex organisms. However, regulation of mammalian astrocyte precursor proliferation during development remains poorly understood. Here, we used Aldh1L1-GFP to identify two morphologically distinct types of proliferative astrocyte precursors: radial glia (RG) in the ventricular zone and a second cell type we call an 'intermediate astrocyte precursor' (IAP) located in the mantle region of the spinal cord. Astrogenic RG and IAP cells proliferated in a progressive ventral-to-dorsal fashion in a tight window from embryonic day 13.5 until postnatal day 3, which correlated precisely with the pattern of active ERK signalling. Conditional loss of BRAF function using BLBP-cre resulted in a 20% decrease in astrocyte production, whereas expression of activated BRAFV600E resulted in astrocyte hyperproliferation. Interestingly, BRAFV600E mitogenic effects in astrocytes were restricted, in part, by the function of p16INK4A-p19(ARF), which limited the temporal epoch for proliferation. Together, these findings suggest that astrocyte precursor proliferation involves distinct RG and IAP cells; is subjected to temporal and spatial control; and depends in part on BRAF signalling at early stages of mammalian spinal cord development.

Abstract LB-514: Contribution of ADAM17 to Kit ligand ectodomain shedding in high-grade gliomas

Abstract Background Malignant anaplastic astrocytoma and glioblastoma are considered to derive from astrocytic precursor cells. They display high vascularization, aggressive growth and invasion into the surrounding brain tissue, and respond poorly to conventional treatments. A disintegrin and metalloproteinases (ADAMs) are membrane-associated metalloproteinases with a complex multi-domain structure. Kit ligand (Kitl), also referred to as stem cell factor (SCF) is synthesized as a membrane-anchored precursor that can be processed to produce the soluble growth factor. As a ligand for Kit receptor tyrosine kinase, Kitl plays important roles in activation of various signalling pathways leading to cell proliferation, differentiation, migration and survival. Previous data has shown that ADAM17 affects Kitl shedding. Hypoxia-induced ADAM17 has been reported to contribute to glioma cell invasion whereas Kitl expression has been demonstrated in glioma cell lines but its function remains unclear. As invasive behaviour is a hallmark of high-grade gliomas, the main aim of our recent study is to investigate the functional role and contribution of ADAM17 to ectodomain shedding of Kitl in these tumors. Methods Quantitative RT-PCR analyses for ADAM17 and Kitl were performed using 36 adult glioma tumor samples including 9 pilocytic astrocytomas WHO grade I (PA I), 9 diffuse astrocytomas WHO grade II (DA II), 9 anaplastic astrocytomas WHO grade III (AA III) and 9 glioblastoma multiforme WHO grade IV (GBM) as well as three (LN-229, LN-308 and U87-MG) glioma cell lines. ADAM17 endogenous expression in glioma cell lines was knocked-down using ADAM17-specific short hairpin RNA (shRNA). The phenotype of the stable transfectants was analyzed by proliferation and migration assays. Immunoblotting and ELISA were performed to detect the Kitl ectodomain released in the cell supernatants. Results We found that ADAM17 and KITLG are overexpressed in high-grade gliomas when compared with low-grade gliomas and normal brain tissue and the higher mRNA levels correlated with the tumor grade. Knock-down of ADAM17 by shRNA significantly reduced glioma cell migration. Moreover shedding of Kitl from control cells (scrambled transfected cells) can be stimulated by addition of phorbol 12-myristate 13-acetate (PMA) together with Ionomycin for 1 hour. In contrast, the PMA/Ionomycin-stimulated Kitl shedding was completely prevented in ADAM17-knock-down cells indicating that ADAM17 is an important PMA/Ionomycin -responsive Kitl sheddase in glioma cells. These results provided evidence for an involvement of ADAM17 in PMA/Ionomycin stimulation of Kitl shedding in glioma cells. Conclusion Our results suggest that ADAM17 in high-grade gliomas effects Kitl ectodomain shedding and may substantially contribute to glioma cell migration. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-514. doi:1538-7445.AM2012-LB-514

Acetaminophen induces oligodendrocyte precursor cell death and reactive astroglia in a primary mixed glial cell culture

Brain damage in acetaminophen (AP) overdose is associated with liver failure but its direct effects has not been evaluated.AimTo study the effect of toxic and non‐toxic doses of AP on astrocyte and oligodendrocyte precursor cell (OPC) viability in a primary mixed glial cell culture.MYMPrimary cultures of mixed glial cells were carried out according to McCarthy and de Vellis (1980). Cells were treated with vehicle (DMSO) or AP (1 and 20 mM). After 48 hs cell viability was assessed; astrocyte marker (GFAP) and OPC marker (PDGFR‐alpha) expression was evaluated by western‐blot (WB) and immunocytochemistry.ResultsThe 1 mM dose of AP did not produce significant differences in cell viability, while 20 mM exposure decreased in 40% cell viability (P<0.001). The 20 mM dose produced the death of the 90% of the OPC in the culture and a decrease of 89% of PDGFR‐alpha expression (P<0.001) with an increase in GFAP optical density/cell and WB expression (47% and 97% respectively) (P<0.05). The 1mM dose did not produce significant changes in GFAP but produce a 25% significant decreased in PDGFR‐alpha expression (p<0.05).ConclusionAP produces direct toxic effects on glial cells characterized by oligodendrocyte death and the induction of reactive astroglia.

Astrocytes as a Source for Extracellular Matrix Molecules and Cytokines

Research of the past 25 years has shown that astrocytes do more than participating and building up the blood-brain barrier and detoxify the active synapse by reuptake of neurotransmitters and ions. Indeed, astrocytes express neurotransmitter receptors and, as a consequence, respond to stimuli. Within the tripartite synapse, the astrocytes owe more and more importance. Besides the functional aspects the differentiation of astrocytes has gained a more intensive focus. Deeper knowledge of the differentiation processes during development of the central nervous system might help explaining and even help treating neurological diseases like Alzheimer’s disease, Amyotrophic lateral sclerosis, Parkinsons disease, and psychiatric disorders in which astrocytes have been shown to play a role. Specific differentiation of neural stem cells toward the astroglial lineage is performed as a multi-step process. Astrocytes and oligodendrocytes develop from a multipotent stem cell that prior to this has produced primarily neuronal precursor cells. This switch toward the more astroglial differentiation is regulated by a change in receptor composition on the cell surface and responsiveness to Fibroblast growth factor and Epidermal growth factor (EGF). The glial precursor cell is driven into the astroglial direction by signaling molecules like Ciliary neurotrophic factor, Bone Morphogenetic Proteins, and EGF. However, the early astrocytes influence their environment not only by releasing and responding to diverse soluble factors but also express a wide range of extracellular matrix (ECM) molecules, in particular proteoglycans of the lectican family and tenascins. Lately these ECM molecules have been shown to participate in glial development. In this regard, especially the matrix protein Tenascin C (Tnc) proved to be an important regulator of astrocyte precursor cell proliferation and migration during spinal cord development. Nevertheless, ECM molecules expressed by reactive astrocytes are also known to act mostly in an inhibitory fashion under pathophysiological conditions. Thus, we further summarize resent data concerning the role of chondroitin sulfate proteoglycans and Tnc under pathological conditions.

Endogenous erythropoietin from astrocyte protects the oligodendrocyte precursor cell against hypoxic and reoxygenation injury

The hypoxia-responsive cytokine erythropoietin (EPO) provides neuroprotective effects in the damaged brain during ischemic events and neurodegenerative diseases. The purpose of the present study is to evaluate the EPO/EPO receptor (EPOR) endogenous system between astrocyte and oligodendrocyte precursor cell (OPC) under hypoxia. We report here elevated EPO mRNA levels and protein release in cultured astrocytes following hypoxic stimulation by quantitative RT-PCR and ELISA. Furthermore, the EPOR gene expressions were detected in cultured OPCs as in astrocytes and microglias by quantitative RT-PCR. Cell staining revealed the EPOR expression in OPC. To evaluate the protective effect of endogenous EPO from astrocyte to OPCs, EPO/EPOR signaling was blocked by EPO siRNA or EPOR siRNA gene silencing in in vitro study. The suppression of endogenous EPO production in astrocytes by EPO siRNA decreased the protection to OPCs against hypoxic stress. Furthermore, OPC with EPOR siRNA had less cell survival after hypoxic/reoxygenation injury. This suggested that EPO/EPOR signaling from astrocyte to OPC could prevent OPC damage under hypoxic/reoxygenation condition. Our present finding of an interaction between astrocytes and OPCs may lead to a new therapeutic approach to OPCs for use against cellular stress and injury.

CD44-Positive Cells Are Candidates for Astrocyte Precursor Cells in Developing Mouse Cerebellum

Neural stem cells are generally considered to be committed to becoming precursor cells before terminally differentiating into either neurons or glial cells during neural development. Neuronal and oligodendrocyte precursor cells have been identified in several areas in the murine central nervous system. The presence of astrocyte precursor cells (APCs) is not so well understood. The present study provides several lines of evidence that CD44-positive cells are APCs in the early postnatal mouse cerebellum. In developing mouse cerebellum, CD44-positive cells, mostly located in the white matter, were positive for the markers of the astrocyte lineage, but negative for the markers of mature astrocytes. CD44-positive cells were purified from postnatal cerebellum by fluorescence-activated cell sorting and characterized in vitro. In the absence of any signaling molecule, many cells died by apoptosis. The surviving cells gradually expressed glial fibrillary acidic protein, a marker for mature astrocytes, indicating that differentiation into mature astrocytes is the default program for these cells. The cells produced no neurospheres nor neurons nor oligodendrocytes under any condition examined, indicating these cells are not neural stem cells. Leukemia inhibitory factor greatly promoted astrocytic differentiation of CD44-positive cells, whereas bone morphogenetic protein 4 (BMP4) did not. Fibroblast growth factor-2 was a potent mitogen for these cells, but was insufficient for survival. BMP4 inhibited activation of caspase-3 and greatly promoted survival, suggesting a novel role for BMP4 in the control of development of astrocytes in cerebellum. We isolated and characterized only CD44 strongly positive large cells and discarded small and/or CD44 weakly positive cells in this study. Further studies are necessary to characterize these cells to help determine whether CD44 is a selective and specific marker for APCs in the developing mouse cerebellum. In conclusion, we succeeded in preparing APC candidates from developing mouse cerebellum, characterized them in vitro, and found that BMPs are survival factors for these cells.

A Developmental Taxonomy of Glioblastoma Defined and Maintained by MicroRNAs

mRNA expression profiling has suggested the existence of multiple glioblastoma subclasses, but their number and characteristics vary among studies and the etiology underlying their development is unclear. In this study, we analyzed 261 microRNA expression profiles from The Cancer Genome Atlas (TCGA), identifying five clinically and genetically distinct subclasses of glioblastoma that each related to a different neural precursor cell type. These microRNA-based glioblastoma subclasses displayed microRNA and mRNA expression signatures resembling those of radial glia, oligoneuronal precursors, neuronal precursors, neuroepithelial/neural crest precursors, or astrocyte precursors. Each subclass was determined to be genetically distinct, based on the significant differences they displayed in terms of patient race, age, treatment response, and survival. We also identified several microRNAs as potent regulators of subclass-specific gene expression networks in glioblastoma. Foremost among these is miR-9, which suppresses mesenchymal differentiation in glioblastoma by downregulating expression of JAK kinases and inhibiting activation of STAT3. Our findings suggest that microRNAs are important determinants of glioblastoma subclasses through their ability to regulate developmental growth and differentiation programs in several transformed neural precursor cell types. Taken together, our results define developmental microRNA expression signatures that both characterize and contribute to the phenotypic diversity of glioblastoma subclasses, thereby providing an expanded framework for understanding the pathogenesis of glioblastoma in a human neurodevelopmental context.

Identity, Fate and Potential of Cells Grown as Neurospheres: Species Matters

It is commonly accepted that adult neurogenesis and gliogenesis follow the same principles through the mammalian class. However, it has been reported that neurogenesis might differ between species, even from the same order, like in rodents. Currently, it is not known if neural stem/progenitor cells (NSPCs) from various species differ in their cell identity and potential. NSPCs can be expanded ex vivo as neurospheres (NSph), a model widely used to study neurogenesis in vitro. Here we demonstrate that rat (r) and mouse (m) NSph display different cell identities, differentiation fate, electrophysiological function and tumorigenic potential. Adult rNSph consist mainly of oligodendroglial progenitors (OPCs), which after repeated passaging proliferate independent of mitogens, whereas adult mNSph show astroglial precursor-like characteristics and retain their mitogen dependency. Most of the cells in rNSph express OPC markers and spontaneously differentiate into oligodendrocytes after growth factor withdrawal. Electrophysiological analysis confirmed OPC characteristics. mNSph have different electrophysiological properties, they express astrocyte precursor markers and spontaneously differentiate primarily into astrocytes. Furthermore, rNSph have the potential to differentiate into oligodendrocytes and astrocytes, whereas mNSph are restricted to the astrocytic lineage. The phenotypic differences between rNSph and mNSph were not due to a distinct response to species specific derived growth factors and are probably not caused by autocrine mechanisms. Our findings suggest that NSph derived from adult rat and mouse brains display different cell identities. Thus, results urge for caution when data derived from NSph are extrapolated to other species or to the in vivo situation, especially when aimed towards the clinical use of human NSph.

Cytoskeletal and nuclear (“nucleoskeletal”) intermediate filaments and disease

Molecular Biology of the CellVol. 22, No. 6 ASCB Annual Meeting HighlightsFree AccessCytoskeletal and nuclear (“nucleoskeletal”) intermediate filaments and diseaseKatherine L. WilsonKatherine L. WilsonJohns Hopkins School of Medicine, Baltimore, MD 21205Search for more papers by this authorPublished Online:13 Oct 2017https://doi.org/10.1091/mbc.e10-12-0960AboutSectionsView PDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmail Cochaired by Ueli Aebi (University of Basel) and Katherine L. Wilson (Johns Hopkins School of Medicine)Why did so many scientists attend the Intermediate Filaments (IFs) Minisymposium on December 15? Two reasons are new connections (IFs can connect to one or both sides of nuclear envelope–spanning LINC complexes) and new evidence that IFs are active players in cell regulation and signaling. Omar Skalli (University of Memphis) discussed synemin, which is highly expressed in astrocyte precursor cells and in astrocytoma cancers. Synemin binds α-actinin and colocalizes with α-actinin at the leading edge of astrocytoma cells. Synemin also associates with the cell cycle regulator Akt. In synemin–down-regulated cells, there is less F-actin at the leading edge, and Akt becomes mislocalized and underactive. Ryan Hobbs (Northwestern University, Chicago) showed evidence that desmoplakin, which links keratin IFs to desmosomes, is aberrantly sequestered on keratin IFs in cells deficient for the SERCA2 calcium pump, which is often defective in Darier’s disease keratinocytes. Stimulation of protein kinase Cα (PKCα) signaling restored proper desmoplakin–keratin association and rescued the weakened intercellular adhesion caused by SERCA2-deficiency, raising the possibility that PKC agonists might be used to treat skin blisters in Darier’s disease patients. Harald Herrmann (German Cancer Research Center, Heidelberg) is using TIRF microscopy to follow the rapid assembly of long keratin filaments in vitro and monitoring the assembly and localization of normal keratins versus disease variants in keratinocytes from mice lacking the entire keratin family. Elisabeth Booth-Gauthier (Carnegie Mellon University) reported that shear forces on cells caused a dramatic shift in the organization of lamin A filaments and widespread changes in chromatin organization within the nucleus as measured by the positions of fiducial marks. These effects were not detected in nuclei made abnormally stiff by expression of progerin, the dominant mutated lamin A protein that causes Hutchinson–Gilford progeria syndrome. This failure to remodel chromatin organization in response to force is proposed as a new mechanotransduction deficiency in accelerated aging. George Dialynas (University of Iowa) discussed a new partnership with University of Iowa clinicians in which novel LMNA mutations that cause Emery–Dreifuss muscular dystrophy are studied in Drosophila muscle to understand how A-type lamins function in vivo. They detect many tissue-specific nuclear defects that provide a potential basis for therapeutic screens. The final speaker, Jason Berk (Johns Hopkins School of Medicine), discussed the posttranslational regulation of both cytoskeletal and nucleoskeletal IFs and a conserved lamin-binding nuclear membrane protein named emerin, by a small sugar, O-GlcNAc, which is added to Ser/Thr residues. Even though O-GlcNAc levels generally decrease during mitosis, emerin is heavily modified by both O-GlcNAcylation and phosphorylation at different stages of mitosis, suggesting emerin is specifically regulated and may have active roles during mitosis. Cochair Katherine Wilson made a plea to recognize the term “nucleoskeleton” as a concept and use nucleoskeleton as a keyword, to enable proper annotation of findings about lamin IFs and other components (e.g., actin, myosins, spectrin, and titin) that contribute to nuclear structure.FOOTNOTESMolecular Biology of the Cell Volume 22 Page 725MBoC is pleased to publish this summary of the Minisymposium “Cytoskeletal and Nuclear Intermediate filaments and Disease” held at the American Society for Cell Biology 2010 Annual Meeting, Philadelphia, PA, December 15, 2010.FiguresReferencesRelatedDetailsCited byThe Z-Disk Final Common Pathway in Cardiomyopathies27 October 2021Farnesyltransferase inhibitor treatment restores chromosome territory positions and active chromosome dynamics in Hutchinson-Gilford progeria syndrome cellsGenome Biology, Vol. 12, No. 8 Vol. 22, No. 6 March 15, 2011715-905 Metrics Downloads & Citations Downloads: 32Citations: 2 History Information© 2011 Wilson. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).PDF download

Astrocyte precursor response to embryonic brain injury

Penetrating traumatic insult during pregnancy is a leading cause of human fetal demise; in particular, trauma to the brain may lead to devastating long-term cognitive sequelae. Perinatal brain injury involves glial precursors, but the neural mechanisms controlling astrocyte ontogeny after injury remain incompletely understood, partly due to a lack of appropriate markers and animal models. We analyzed astrocyte precursor response to injury at the beginning (E11) and peak (E15) of gliogenesis in an avian tectal model of penetrating embryonic brain trauma, without confounding maternal and sibling effects. At both ages, lateral ventricular dilatation, necrotic foci, periventricular cysts and intraventricular hemorrhages were observed distal to stab wounds two days after a unilateral stab injury to optic tecta. Neuronal (TUBB3) and oligodendrocyte precursor (PLP) markers were down-regulated, even far-removed from the wound site. In contrast, the mature astrocyte marker, GFAP, was up-regulated at the wound site, around necrotic areas and cysts, plus in usual areas of GFAP expression. Increased inflammatory response and apoptotic cell death were also confirmed in the injured tecta. Increased expression of NFIA, SOX9 and GLAST at the wound site and in the ventricular zone (VZ) of the injured tecta indicated an astroglial precursor response. However, cell division increased in the VZ only in early (E11) injury, but not later (E15), indicating that in late injury the astrogliogenesis occurring after acute injury is predominantly due to precursor differentiation rather than precursor proliferation. The inability to replenish the glial precursor pool during the critical period of vulnerability to injury may be an important cause of subsequent developmental abnormalities.

Neuronal hyperexcitability and seizures are associated with changes in glial–neuronal interactions in the hippocampus of a mouse model of epilepsy with mental retardation

Hippocampal excitability and the metabolic glial-neuronal interactions were investigated in 22-week-old mice with motor neuron degeneration (mnd), a model of progressive epilepsy with mental retardation. Mnd mice developed spontaneous spikes in the hippocampus and were more susceptible to kainate-induced seizures compared with control mice. Neuronal hyperexcitability in their hippocampus was confirmed by the selective increase of c-Fos positive nuclei. Glial activation and pro-inflammatory cytokines over-expression were observed in the hippocampus of mnd mice, even in the absence of marked hippocampal neurodegeneration, as suggested by unchanged amounts of neuroactive amino acids and N-acetyl aspartate. Concentration of other amino acids, including GABA and glutamate, was not changed as well. However, ex vivo(13) C magnetic resonance spectroscopy, after simultaneous injection of [1-(13) C]glucose and [1,2-(13) C]acetate, followed by decapitation, showed decreased [1,2-(13) C]GABA formation from hippocampal astrocytic precursors and a marked reduction in [4,5-(13) C]glutamate derived from glutamine. We suggest that astrocyte dysfunction plays a primary role in the pathology and that mnd mice are of value to investigate early pathogenetic mechanism of progressive epilepsy with mental retardation.

Cryopreservation of Cortical Tissue Blocks for the Generation of Highly Enriched Neuronal Cultures

In this study, we outline a standardized protocol for the successful cryopreservation and thawing of cortical brain tissue blocks to generate highly enriched neuronal cultures. For this protocol the freezing medium used is 10% dimethyl sulfoxide (DMSO) diluted in Hank's Buffered Salt Solution (HBSS). Blocks of cortical tissue are transferred to cryovials containing the freezing medium and slowly frozen at -1°C/min in a rate-controlled freezing container. Post-thaw processing and dissociation of frozen tissue blocks consistently produced neuronal-enriched cultures which exhibited rapid neuritic growth during the first 5 days in culture and significant expansion of the neuronal network within 10 days. Immunocytochemical staining with the astrocytic marker glial fibrillary acidic protein (GFAP) and the neuronal marker beta-tubulin class III, revealed high numbers of neurons and astrocytes in the cultures. Generation of neural precursor cell cultures after tissue block dissociation resulted in rapidly expanding neurospheres, which produced large numbers of neurons and astrocytes under differentiating conditions. This simple cryopreservation protocol allows for the rapid, efficient, and inexpensive preservation of cortical brain tissue blocks, which grants increased flexibility for later generation of neuronal, astrocyte, and neuronal precursor cell cultures.

Cryopreservation of Cortical Tissue Blocks for the Generation of Highly Enriched Neuronal Cultures

In this study, we outline a standardized protocol for the successful cryopreservation and thawing of cortical brain tissue blocks to generate highly enriched neuronal cultures. For this protocol the freezing medium used is 10% dimethyl sulfoxide (DMSO) diluted in Hank's Buffered Salt Solution (HBSS). Blocks of cortical tissue are transferred to cryovials containing the freezing medium and slowly frozen at -1°C/min in a rate-controlled freezing container. Post-thaw processing and dissociation of frozen tissue blocks consistently produced neuronal-enriched cultures which exhibited rapid neuritic growth during the first 5 days in culture and significant expansion of the neuronal network within 10 days. Immunocytochemical staining with the astrocytic marker glial fibrillary acidic protein (GFAP) and the neuronal marker beta-tubulin class III, revealed high numbers of neurons and astrocytes in the cultures. Generation of neural precursor cell cultures after tissue block dissociation resulted in rapidly expanding neurospheres, which produced large numbers of neurons and astrocytes under differentiating conditions. This simple cryopreservation protocol allows for the rapid, efficient, and inexpensive preservation of cortical brain tissue blocks, which grants increased flexibility for later generation of neuronal, astrocyte, and neuronal precursor cell cultures.

Effect of 6-hydroxydopamine (6-OHDA) on proliferation of glial cells in the rat cortex and striatum: evidence for de-differentiation of resident astrocytes

Reactive astrogliosis is the universal response to any brain insult. It is characterized by cellular hypertrophy, up-regulation of the astrocyte marker glial fibrillary acidic protein (GFAP), and proliferation. The source of these proliferating cells is under intense debate. Progenitor cells derived from the subventricular zone (SVZ), cells positive for chondroitin sulfate proteoglycan (NG2(+)), and de-differentiated astrocytes have been proposed as the origin of proliferating cells following injury. We have analyzed the effect of intraventricular-applied 6-hydroxydopamine (6-OHDA) on the proliferation and morphology of astrocytes in rat cortex and striatum by means of immunohistochemistry and confocal laser microscopy. At 4 days post-lesion, GFAP expression increased markedly. A subpopulation of the GFAP(+) cells co-expressed Ki-67, indicating that these cells were proliferating. To investigate whether these cells (1) arose from migrating SVZ progenitor cells, (2) derived from NG2(+) progenitor cells, or (3) de-differentiated from resident astrocytes, we studied the expression of the migration marker doublecortin (Dcx), the oligodendrocyte progenitor marker NG2, and the progenitor markers Nestin and Pax6. The proliferating Ki-67(+) cells co-expressed Nestin and Pax6, whereas no co-expression of Ki-67 with NG2 or the migration marker Dcx was observed. Thus, resident astrocytes de-differentiate, in response to the intraventricular application of 6-OHDA, to a phenotype resembling radial glia cells, which represent transient astrocyte precursors during development. An understanding of the mechanisms of the de-differentiation of mature astrocytes might be useful for designing new approaches to cell therapy in neurodegenerative diseases such as Parkinson's disease.

Role of BMPs in controlling the spatial and temporal origin of GFAP astrocytes in the embryonic spinal cord

In the vertebrate central nervous system (CNS), astrocytes are the most abundant and functionally diverse glial cell population. However, the mechanisms underlying their specification and differentiation are still poorly understood. In this study, we have defined spatially and temporally the origin of astrocytes and studied the role of BMPs in astrocyte development in the embryonic chick spinal cord. Using explant cultures, we show that astrocyte precursors started migrating out of the neuroepithelium in the mantle layer from E5, and that the dorsal-most level of the neuroepithelium, from the roof plate to the dl3 level, did not generate GFAP-positive astrocytes. Using a variety of early astrocyte markers together with functional analyses, we show that dorsal-most progenitors displayed a potential for astrocyte production but that dorsally-derived BMP signalling, possibly mediated through BMP receptor 1B, promoted neuronal specification instead. BMP treatment completely prevented astrocyte development from intermediate spinal cord explants at E5, whereas it promoted it at E6. Such an abrupt change in the response of this tissue to BMP signalling could be correlated to the onset of new foci of BMP activity and enhanced expression of BMP receptor 1A, suggesting that BMP signalling could promote astrocyte development in this region.

GFAPδ in radial glia and subventricular zone progenitors in the developing human cortex

A subpopulation of glial fibrillary acidic protein (GFAP)-expressing cells located along the length of the lateral ventricles in the subventricular zone (SVZ) have been identified as the multipotent neural stem cells of the adult mammalian brain. We have previously found that, in the adult human brain, a splice variant of GFAP, termed GFAPdelta, was expressed specifically in these cells. To investigate whether GFAPdelta is also present in the precursors of SVZ astrocytes during development and whether GFAPdelta could play a role in the developmental process, we analyzed GFAPdelta expression in the normal developing human cortex and in the cortex of foetuses with the migration disorder lissencephaly type II. We demonstrated for the first time that GFAPdelta is specifically expressed in radial glia and SVZ neural progenitors during human brain development. Expression of GFAPdelta in radial glia starts at around 13 weeks of pregnancy and disappears before birth. GFAPdelta is continuously expressed in the SVZ progenitors at later gestational ages and in the postnatal brain. Co-localization with Ki67 proved that these GFAPdelta-expressing cells are able to proliferate. Furthermore, we showed that the expression pattern of GFAPdelta was disturbed in lissencephaly type II. Overall, these results suggest that the adult SVZ is indeed a remnant of the foetal SVZ, which develops from radial glia. Furthermore, we provide evidence that GFAPdelta can distinguish resting astrocytes from proliferating SVZ progenitors.

BMP7 and SHH regulate Pax2 in mouse retinal astrocytes by relieving TLX repression

Pax2 is essential for development of the neural tube, urogenital system, optic vesicle, optic cup and optic tract. In the eye, Pax2 deficiency is associated with coloboma, a loss of astrocytes in the optic nerve and retina, and abnormal axonal pathfinding of the ganglion cell axons at the optic chiasm. Thus, appropriate expression of Pax2 is essential for astrocyte determination and differentiation. Although BMP7 and SHH have been shown to regulate Pax2 expression, the molecular mechanism by which this regulation occurs is not well understood. In this study, we determined that BMP7 and SHH activate Pax2 expression in mouse retinal astrocyte precursors in vitro. SHH appeared to play a dual role in Pax2 regulation; 1) SHH may regulate BMP7 expression, and 2) the SHH pathway cooperates with the BMP pathway to regulate Pax2 expression. BMP and SHH pathway members can interact separately or together with TLX, a repressor protein in the tailless transcription factor family. Here we show that the interaction of both pathways with TLX relieves the repression of Pax2 expression in mouse retinal astrocytes. Together these data reveal a new mechanism for the cooperative actions of signaling pathways in astrocyte determination and differentiation and suggest interactions of regulatory pathways that are applicable to other developmental programs.