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Revealing the molecular events associated with reprogramming different somatic cell types to pluripotency is critical for understanding the characteristics of induced pluripotent stem cell (iPSC) therapeutic derivatives. Inducible reprogramming factor transgenic cells or animals-designated as secondary (2°) reprogramming systems-not only provide excellent experimental tools for such studies but also offer a strategy to study the variances in cellular reprogramming outcomes due to different in vitro and in vivo environments. To make such studies less cumbersome, it is desirable to have a variety of efficient reprogrammable mouse systems to induce successful mass reprogramming in somatic cell types. Here, we report the development of two transgenic mouse lines from which 2° cells reprogram with unprecedented efficiency. These systems were derived by exposing primary reprogramming cells containing doxycycline-inducible Yamanaka factor expression to a transient interruption in transgene expression, resulting in selection for a subset of clones with robust transgene response. These systems also include reporter genes enabling easy readout of endogenous Oct4 activation (GFP), indicative of pluripotency, and reprogramming transgene expression (mCherry). Notably, somatic cells derived from various fetal and adult tissues from these 2° mouse lines gave rise to highly efficient and rapid reprogramming, with transgene-independent iPSC colonies emerging as early as 1 wk after induction. These mouse lines serve as a powerful tool to explore sources of variability in reprogramming and the mechanistic underpinnings of efficient reprogramming systems.
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Reprogramação Celular , Doxiciclina , Animais , Camundongos , Camundongos Transgênicos , Reprogramação Celular/genética , Transgenes , Células Clonais , Doxiciclina/farmacologiaRESUMO
Astrocytes that reside in superficial (SL) and deep cortical layers have distinct molecular profiles and morphologies, which may underlie specific functions. Here, we demonstrate that the production of SL and deep layer (DL) astrocyte populations from neural progenitor cells in the mouse is temporally regulated. Lineage tracking following in utero and postnatal electroporation with PiggyBac (PB) EGFP and birth dating with EdU and FlashTag, showed that apical progenitors produce astrocytes during late embryogenesis (E16.5) that are biased to the SL, while postnatally labeled (P0) astrocytes are biased to the DL. In contrast, astrocytes born during the predominantly neurogenic window (E14.5) showed a random distribution in the SL and DL. Of interest, E13.5 astrocytes birth dated at E13.5 with EdU showed a lower layer bias, while FT labeling of apical progenitors showed no bias. Finally, examination of the morphologies of "biased" E16.5- and P0-labeled astrocytes demonstrated that E16.5-labeled astrocytes exhibit different morphologies in different layers, while P0-labeled astrocytes do not. Differences based on time of birth are also observed in the molecular profiles of E16.5 versus P0-labeled astrocytes. Altogether, these results suggest that the morphological, molecular, and positional diversity of cortical astrocytes is related to their time of birth from ventricular/subventricular zone progenitors.
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Astrócitos , Córtex Cerebral , Células-Tronco Neurais , Animais , Astrócitos/metabolismo , Astrócitos/citologia , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/citologia , Camundongos , Camundongos Transgênicos , Feminino , Animais Recém-Nascidos , Regulação da Expressão Gênica no Desenvolvimento , Transcriptoma , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Ventrículos Cerebrais/citologia , Camundongos Endogâmicos C57BLRESUMO
Proteome profiles of precious tissue samples have great clinical potential for accelerating disease biomarker discovery and promoting novel strategies for early diagnosis and treatment. However, tiny clinical tissue samples are often difficult to handle and analyze with conventional proteomic methods. Automated digital microfluidic (DMF) workflows facilitate the manipulation of size-limited tissue samples. Here, we report the assessment of a DMF microproteomics workflow enabled by a photocleavable surfactant for proteomic analysis of minute tissue samples. The surfactant 4-hexylphenylazosulfonate (Azo) was found to facilitate fast droplet movement on DMF and enhance the proteomics analysis. Comparisons of Azo and n-Dodecyl ß-d-maltoside (DDM) using small samples of HeLa digest standards and MCF-7 cell digests revealed distinct differences at the peptide level despite similar results at the protein level. The DMF microproteomics workflow was applied for the sample preparation of â¼3 µg biopsies from murine brain tissue. A total of 1969 proteins were identified in three samples, including established neural biomarkers and proteins related to synaptic signaling. Going forward, we propose that the Azo-enabled DMF workflow has the potential to advance the practical clinical application of DMF for the analysis of size-limited tissue samples.
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Direct lineage reprogramming is the conversion of one specialized cell type to another without the need for a pluripotent intermediate. To date, a wide variety of cell types have been successfully generated using direct reprogramming, both in vitro and in vivo. These newly converted cells have the potential to replace cells that are lost to disease and/or injury. In this chapter, we will focus on direct reprogramming in the central nervous system. We will review current progress in the field with regards to all the major neural cell types and explore how cellular heterogeneity, both in the starter cell and target cell population, may have implications for direct reprogramming. Finally, we will discuss new technologies that will improve our understanding of the reprogramming process and aid the development of more specific and efficient future CNS-based reprogramming strategies.
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Linhagem da Célula , Reprogramação Celular , Sistema Nervoso Central/citologia , Neurônios/citologia , Diferenciação Celular , HumanosRESUMO
Astrocytes, a type of glial cell in the central nervous system (CNS), adopt diverse states in response to injury that are influenced by their location relative to the insult. Here, we describe a platform for spatially resolved, single-cell transcriptomics and proteomics, called tDISCO (tissue-digital microfluidic isolation of single cells for -Omics). We use tDISCO alongside two high-throughput platforms for spatial (Visium) and single-cell transcriptomics (10X Chromium) to examine the heterogeneity of the astrocyte response to a cortical ischemic stroke in male mice. We show that integration of Visium and 10X Chromium datasets infers two astrocyte populations, proximal or distal to the injury site, while tDISCO determines the spatial boundaries and molecular profiles that define these populations. We find that proximal astrocytes show differences in lipid shuttling, with enriched expression of Apoe and Fabp5. Our datasets provide a resource for understanding the roles of astrocytes in stroke and showcase the utility of tDISCO for hypothesis-driven, spatially resolved single-cell experiments.
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Astrócitos , Acidente Vascular Cerebral , Camundongos , Animais , Masculino , Astrócitos/metabolismo , Sistema Nervoso Central/metabolismo , Acidente Vascular Cerebral/genética , Acidente Vascular Cerebral/metabolismo , Perfilação da Expressão Gênica , Cromo/metabolismoRESUMO
Adult neurogenesis is regulated by a number of cellular players within the neurogenic niche. Astrocytes participate actively in brain development, regulation of the mature central nervous system (CNS), and brain plasticity. They are important regulators of the local environment in adult neurogenic niches through the secretion of diffusible morphogenic factors, such as Wnts. Astrocytes control the neurogenic niche also through membrane-associated factors, however, the identity of these factors and the mechanisms involved are largely unknown. In this study, we sought to determine the mechanisms underlying our earlier finding of increased neuronal differentiation of neural progenitor cells when cocultured with astrocytes lacking glial fibrillary acidic protein (GFAP) and vimentin (GFAP(-/-) Vim(-/-) ). We used primary astrocyte and neurosphere cocultures to demonstrate that astrocytes inhibit neuronal differentiation through a cell-cell contact. GFAP(-/-) Vim(-/-) astrocytes showed reduced endocytosis of Notch ligand Jagged1, reduced Notch signaling, and increased neuronal differentiation of neurosphere cultures. This effect of GFAP(-/-) Vim(-/-) astrocytes was abrogated in the presence of immobilized Jagged1 in a manner dependent on the activity of γ-secretase. Finally, we used GFAP(-/-) Vim(-/-) mice to show that in the absence of GFAP and vimentin, hippocampal neurogenesis under basal conditions as well as after injury is increased. We conclude that astrocytes negatively regulate neurogenesis through the Notch pathway, and endocytosis of Notch ligand Jagged1 in astrocytes and Notch signaling from astrocytes to neural stem/progenitor cells depends on the intermediate filament proteins GFAP and vimentin.
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Astrócitos/metabolismo , Proteínas de Ligação ao Cálcio/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/genética , Neurogênese/genética , Receptores Notch/genética , Vimentina/genética , Secretases da Proteína Precursora do Amiloide/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Astrócitos/citologia , Proteínas de Ligação ao Cálcio/metabolismo , Comunicação Celular/genética , Diferenciação Celular , Técnicas de Cocultura , Endocitose , Regulação da Expressão Gênica no Desenvolvimento , Proteína Glial Fibrilar Ácida , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteína Jagged-1 , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/deficiência , Cultura Primária de Células , Receptores Notch/metabolismo , Proteínas Serrate-Jagged , Transdução de Sinais , Células-Tronco/citologia , Células-Tronco/metabolismo , Vimentina/deficiência , Proteínas Wnt/genética , Proteínas Wnt/metabolismoRESUMO
Single-cell gene expression levels show substantial variations among cells in seemingly homogenous populations. Astrocytes perform many control and regulatory functions in the central nervous system. In contrast to neurons, we have limited knowledge about functional diversity of astrocytes and its molecular basis. To study astrocyte heterogeneity and stem/progenitor cell properties of astrocytes, we used single-cell gene expression profiling in primary mouse astrocytes and dissociated mouse neurosphere cells. The transcript number variability for astrocytes showed lognormal features and revealed that cells in primary cultures to a large extent co-express markers of astrocytes and neural stem/progenitor cells. We show how subpopulations of cells can be identified at single-cell level using unsupervised algorithms and that gene correlations can be used to identify differences in activity of important transcriptional pathways. We identified two subpopulations of astrocytes with distinct gene expression profiles. One had an expression profile very similar to that of neurosphere cells, whereas the other showed characteristics of activated astrocytes in vivo.
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Astrócitos/classificação , Perfilação da Expressão Gênica/métodos , Análise de Célula Única , Animais , Astrócitos/metabolismo , Células Cultivadas , Expressão Gênica , Camundongos , RNA Mensageiro/análise , Células-Tronco/metabolismo , Regulação para CimaRESUMO
BACKGROUND: In immune-mediated diseases of the central nervous system, astrocytes exposed to interferon-γ (IFN-γ) can express major histocompatibility complex (MHC) class II molecules and antigens on their surface. MHC class II molecules are thought to be delivered to the cell surface by membrane-bound vesicles. However, the characteristics and dynamics of this vesicular traffic are unclear, particularly in reactive astrocytes, which overexpress intermediate filament (IF) proteins that may affect trafficking. The aim of this study was to determine the mobility of MHC class II vesicles in wild-type (WT) astrocytes and in astrocytes devoid of IFs. METHODS: The identity of MHC class II compartments in WT and IF-deficient astrocytes 48 h after IFN-γ activation was determined immunocytochemically by using confocal microscopy. Time-lapse confocal imaging and Alexa Fluor546-dextran labeling of late endosomes/lysosomes in IFN-γ treated cells was used to characterize the motion of MHC class II vesicles. The mobility of vesicles was analyzed using ParticleTR software. RESULTS: Confocal imaging of primary cultures of WT and IF-deficient astrocytes revealed IFN-γ induced MHC class II expression in late endosomes/lysosomes, which were specifically labeled with Alexa Fluor546-conjugated dextran. Live imaging revealed faster movement of dextran-positive vesicles in IFN-γ-treated than in untreated astrocytes. Vesicle mobility was lower in IFN-γ-treated IF-deficient astrocytes than in WT astrocytes. Thus, the IFN-γ-induced increase in the mobility of MHC class II compartments is IF-dependent. CONCLUSIONS: Since reactivity of astrocytes is a hallmark of many CNS pathologies, it is likely that the up-regulation of IFs under such conditions allows a faster and therefore a more efficient delivery of MHC class II molecules to the cell surface. In vivo, such regulatory mechanisms may enable antigen-presenting reactive astrocytes to respond rapidly and in a controlled manner to CNS inflammation.
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Astrócitos/metabolismo , Compartimento Celular/fisiologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Interferon gama/fisiologia , Proteínas de Filamentos Intermediários/fisiologia , Animais , Células Cultivadas , Interferon gama/genética , Proteínas de Filamentos Intermediários/genética , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transporte Proteico/genética , Transporte Proteico/fisiologia , Regulação para Cima/genética , Regulação para Cima/fisiologiaRESUMO
Background: The use of animals and animal-derived products in ethnopharmacological applications is an ancient human practice that continues in many regions today. The local people of the Himalayan region harbor rich traditional knowledge used to treat a variety of human ailments. The present study was intended with the aim of examining animal-based traditional medicine utilized by the population of the Himalayan region of Azad Jammu and Kashmir. Methods: Data were collected from 2017 to 2019 through individual and group interviews. Data on traditional uses of animal products were analyzed, utilizing following indices such as the frequency of citation, use value, relative importance, similarity index, principal component analysis, and cluster analysis to find the highly preferred species in the area. Results: Ethnomedicinal uses of 62 species of vertebrates and invertebrates were documented. Flesh, fat, bone, whole body, milk, skin, egg, head, feathers, bile, blood, and honey were all used in these applications. The uses of 25 animals are reported here for the first time from the study area (mainly insects and birds, including iconic species like the kalij pheasant, Lophura leucomelanos; Himalayan monal, L. impejanus; and western tragopon, Tragopan melanocephalus). The diversity and range of animal-based medicines utilized in these communities are indications of their strong connections with local ecosystems. Conclusion: Our results provide baseline data valuable for the conservation of vertebrate and invertebrate diversity in the region of Himalayan of Azad Jammu and Kashmir. It is possible that screening this fauna for medicinally active chemicals could contribute to the development of new animal-based drugs.
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Direct neuronal reprogramming, the process whereby a terminally differentiated cell is converted into an induced neuron without traversing a pluripotent state, has tremendous therapeutic potential for a host of neurodegenerative diseases. While there is strong evidence for astrocyte-to-neuron conversion in vitro, in vivo studies in the adult brain are less supportive or controversial. Here, we set out to enhance the efficacy of neuronal conversion of adult astrocytes in vivo by optimizing the neurogenic capacity of a driver transcription factor encoded by the proneural gene Ascl1. Specifically, we mutated six serine phospho-acceptor sites in Ascl1 to alanines (Ascl1 SA 6) to prevent phosphorylation by proline-directed serine/threonine kinases. Native Ascl1 or Ascl1 SA 6 were expressed in adult, murine cortical astrocytes under the control of a glial fibrillary acidic protein (GFAP) promoter using adeno-associated viruses (AAVs). When targeted to the cerebral cortex in vivo, mCherry+ cells transduced with AAV8-GFAP-Ascl1 SA 6-mCherry or AAV8-GFAP-Ascl1-mCherry expressed neuronal markers within 14 days post-transduction, with Ascl1 SA 6 promoting the formation of more mature dendritic arbors compared to Ascl1. However, mCherry expression disappeared by 2-months post-transduction of the AAV8-GFAP-mCherry control-vector. To circumvent reporter issues, AAV-GFAP-iCre (control) and AAV-GFAP-Ascl1 (or Ascl1 SA 6)-iCre constructs were generated and injected into the cerebral cortex of Rosa reporter mice. In all comparisons of AAV capsids (AAV5 and AAV8), GFAP promoters (long and short), and reporter mice (Rosa-zsGreen and Rosa-tdtomato), Ascl1 SA 6 transduced cells more frequently expressed early- (Dcx) and late- (NeuN) neuronal markers. Furthermore, Ascl1 SA 6 repressed the expression of astrocytic markers Sox9 and GFAP more efficiently than Ascl1. Finally, we co-transduced an AAV expressing ChR2-(H134R)-YFP, an optogenetic actuator. After channelrhodopsin photostimulation, we found that Ascl1 SA 6 co-transduced astrocytes exhibited a significantly faster decay of evoked potentials to baseline, a neuronal feature, when compared to iCre control cells. Taken together, our findings support an enhanced neuronal conversion efficiency of Ascl1 SA 6 vs. Ascl1, and position Ascl1 SA 6 as a critical transcription factor for future studies aimed at converting adult brain astrocytes to mature neurons to treat disease.
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The cerebral cortex develops from dorsal forebrain neuroepithelial progenitor cells. Following the initial expansion of the progenitor cell pool, these cells generate neurons of all the cortical layers and then astrocytes and oligodendrocytes. Yet, the regulatory pathways that control the expansion and maintenance of the progenitor cell pool are currently unknown. Here we define six basic pathway components that regulate proliferation of cortically specified human neuroepithelial stem cells (cNESCs) in vitro without the loss of cerebral cortex developmental potential. We show that activation of FGF and inhibition of BMP and ACTIVIN A signalling are required for long-term cNESC proliferation. We also demonstrate that cNESCs preserve dorsal telencephalon-specific potential when GSK3, AKT and nuclear CATENIN-ß1 activity are low. Remarkably, regulation of these six pathway components supports the clonal expansion of cNESCs. Moreover, cNESCs differentiate into lower- and upper-layer cortical neurons in vitro and in vivo. The identification of mechanisms that drive the neuroepithelial stem cell self-renewal and differentiation and preserve this potential in vitro is key to developing regenerative and cell-based therapeutic approaches to treat neurological conditions.
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Quinase 3 da Glicogênio Sintase , Células Neuroepiteliais , Diferenciação Celular/fisiologia , Córtex Cerebral , Humanos , Células-Tronco , TelencéfaloRESUMO
Mammalian SVZ progenitors continuously generate new neurons in the olfactory bulb. After injury, changes in SVZ cell number suggest injury-induced migration. Studies that trace the migration of SVZ precursors into neurodegenerating areas are lacking. Previously, we showed a decrease in BrdU+SVZ cells following excitotoxic damage to the immature rat cortex. Here, we demonstrate that NMDA-induced injury forces endogenous Cell Tracker Green (CTG) labeled VZ/SVZ precursors out of the SVZ into the neurodegenerating cortex. CTG+/Nestin+/Filamin A+ precursors are closely associated with vimentin+/GFAP+/GLAST+ filaments and express both chemokine receptor CXCR4 and Robo1. In the cortex, SVZ-derived progenitors show a progressive expression of developing, migrating and mature neurons and glial markers. CTG+/GFAP+ astrocytes greatly outnumber CTG+/MAP2+/NeuN+ neurons. SVZ-derived progenitors differentiate into both tbr1+ cortical glutamatergic neurons and calretinin+ interneurons. But, there is little integration of these neurons into the existing circuitry, as seen by Fluorogold retrograde tracing from the internal capsule.
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Movimento Celular/fisiologia , Córtex Motor/patologia , Degeneração Neural/patologia , Córtex Somatossensorial/patologia , Células-Tronco/citologia , Fatores Etários , Animais , Astrócitos/citologia , Biomarcadores/metabolismo , Calbindina 2 , Diferenciação Celular/fisiologia , Divisão Celular/fisiologia , Agonistas de Aminoácidos Excitatórios/toxicidade , Feminino , Gliose/patologia , Cápsula Interna/citologia , Cápsula Interna/crescimento & desenvolvimento , Masculino , Córtex Motor/fisiologia , N-Metilaspartato/toxicidade , Degeneração Neural/fisiopatologia , Proteínas do Tecido Nervoso/metabolismo , Neurônios/citologia , Neurotoxinas/toxicidade , Ratos , Ratos Long-Evans , Receptores CXCR4/metabolismo , Receptores Imunológicos/metabolismo , Proteína G de Ligação ao Cálcio S100/metabolismo , Córtex Somatossensorial/fisiologia , Células-Tronco/metabolismo , Proteínas RoundaboutRESUMO
Precursor cells have been shown to be affected by oxidative stress, in vivo and vitro, but little is known about the expression of antioxidant mechanisms in neuronal/glial differentiation. We have characterized the expression of Cu/Zn superoxide dismutase (Cu/Zn SOD), one of the main antioxidant proteins involved in the breakdown of superoxide, in the immature rat dorsolateral subventricular zone (SVZ), rostral migratory stream (RMS) and hippocampal subgranular zone (SGZ). Progenitor cells were identified immunohistochemically on cryostat sections by 5'Bromodeoxyuridine (BrdU) incorporation and expressing cells were further characterized using double labeling for progenitor markers. In the SVZ, only a subpopulation of BrdU+ cells, mostly found in the medial SVZ, expressed Cu/Zn SOD. These cells were mostly nestin+ and some were also vimentin+. In contrast, in the lateral SVZ few Cu/Zn SOD+/BrdU+ cells were found. These were primarily nestin+, vimentin-, showed some PSA-NCAM expression, but only a few were NG2+. In the RMS and SGZ virtually all BrdU+ progenitors were Cu/Zn SOD+ and expressed nestin and vimentin. Some RMS cells were also PSA-NCAM+. These findings show a heterogeneous expression of Cu/Zn SOD in restricted cell types in the germinative zones and suggest a role for antioxidant Cu/Zn SOD in progenitor cells of the immature rat brain.
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Antioxidantes/metabolismo , Encéfalo/crescimento & desenvolvimento , Neuroglia/enzimologia , Neurônios/enzimologia , Células-Tronco/enzimologia , Superóxido Dismutase/metabolismo , Animais , Animais Recém-Nascidos , Biomarcadores/metabolismo , Encéfalo/citologia , Encéfalo/enzimologia , Bromodesoxiuridina , Diferenciação Celular/fisiologia , Movimento Celular/fisiologia , Proliferação de Células , Proteínas de Filamentos Intermediários/metabolismo , Ventrículos Laterais/citologia , Ventrículos Laterais/crescimento & desenvolvimento , Ventrículos Laterais/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Nestina , Molécula L1 de Adesão de Célula Nervosa/metabolismo , Neuroglia/citologia , Neurônios/citologia , Estresse Oxidativo/fisiologia , Ratos , Ratos Long-Evans , Ácidos Siálicos/metabolismo , Vimentina/metabolismoRESUMO
BACKGROUND: In the nervous system, as in other organs, Cu/Zn superoxide dismutase (Cu/Zn SOD) is a key antioxidant enzyme involved in superoxide detoxification in normal cellular metabolism and after cell injury. Although it has been suggested that immature brain has a different susceptibility to oxidative damage than adult brain, the distribution and cell-specific expression of this enzyme in immature brain and after postnatal brain damage has not been documented. METHODS: In this study, we used immunohistochemistry and western blot to analyze the expression of Cu/Zn SOD in intact immature rat brain and in immature rat brain after an NMDA-induced excitotoxic cortical injury performed at postnatal day 9. Double immunofluorescence labelling was used to identify Cu/Zn SOD-expressing cell populations. RESULTS: In intact immature brain, Cu/Zn SOD enzyme was widely expressed at high levels in neurons mainly located in cortical layers II, III and V, in the sub-plate, in the pyriform cortex, in the hippocampus, and in the hypothalamus. Glial fibrillary acidic protein-positive cells only showed Cu/Zn SOD expression in the glia limitans and in scattered cells of the ventricle walls. No expression was detected in interfascicular oligodendroglia, microglia or endothelial cells. Following excitotoxic damage, neuronal Cu/Zn SOD was rapidly downregulated (over 2-4 hours) at the injection site before neurodegeneration signals and TUNEL staining were observed. Later, from 1 day post-lesion onward, an upregulation of Cu/Zn SOD was found due to increased expression in astroglia. A further increase was observed at 3, 5 and 7 days that corresponded to extensive induction of Cu/Zn SOD in highly reactive astrocytes and in the astroglial scar. CONCLUSION: We show here that, in the intact immature brain, the expression of Cu/Zn SOD was mainly found in neurons. When damage occurs, a strong and very rapid downregulation of this enzyme precedes neuronal degeneration, and is followed by an upregulation of Cu/Zn SOD in astroglial cells.
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BACKGROUND: The forebrain subventricular zone (SVZ)-olfactory bulb pathway and hippocampal subgranular zone (SGZ) generate neurons into adulthood in the mammalian brain. Neurogenesis increases after injury to the adult brain, but few studies examine the effect of injury on neural and glial precursors in the postnatal brain. To characterize the spatio-temporal dynamics of cell proliferation in the germinative zones, this study utilized a model of postnatal damage induced by NMDA injection in the right sensorimotor cortex at postnatal day 9. Dividing cell populations were labeled with 5-Bromodeoxyuridine (BrdU) in the intact and damaged postnatal brain. Identity of proliferating cells was determined by double immunolabeling with nestin, GFAP, NeuN and tomato lectin (TL). RESULTS: In the control brain, grouped BrdU+ cells were observed in the Rostral Migratory Stream (RMS), SVZ and SGZ. Maximal proliferation was seen at P12, persisted until P23 and diminished by P49. After injury, a striking reduction in the number of BrdU+ cells was observed in the ipsilateral SVZ from 10 hours (58% decrease) until 14 days post-lesion (88% decrease). In contrast, an increase in grouped BrdU+ cells was seen in the striatum adjacent to the depleted SVZ. Significantly reduced numbers of BrdU+ cells were also seen in the RMS until 3 days post-lesion. No changes were noted in the SGZ. Both in controls and lesioned hemispheres, BrdU+ cells located in the germinal zones were mostly nestin positive and negative for GFAP, NeuN, and TL. In the SVZ area lining the ventricle, BrdU+/nestin+ cells were mainly located between TL+ ependyma and parenchymal GFAP+ astrocytes. After excitotoxicity, a decrease in the number and orientation of GFAP/nestin+ prolongations leaving the SVZ to the cortex, corpus callosum and striatum was noted until 5 days post-lesion. CONCLUSION: Postnatal excitotoxic injury differentially affects proliferating cells in the germinative zones: no change is observed in the dentate gyrus whereas excitotoxicity causes a significant decrease in proliferating cells in the SVZ and RMS. Depletion of BrdU+ cells in the postnatal SVZ and RMS differs from previous studies after adult brain injury and may affect the SVZ-RMS migration and is suggestive of progenitor recruitment to injured areas.
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Encéfalo/citologia , Encéfalo/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Agonistas de Aminoácidos Excitatórios/toxicidade , Camadas Germinativas/citologia , Camadas Germinativas/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Encéfalo/crescimento & desenvolvimento , Feminino , Camadas Germinativas/fisiologia , Masculino , N-Metilaspartato/toxicidade , Ratos , Ratos Long-EvansRESUMO
Reactive astrocytes (RAs) have been reported to convert to multipotent neural stem cells (NSCs) capable of neurosphere (NS) formation and multilineage differentiation in vitro. Using genetic tagging, we determined that subventricular zone (SVZ) NSCs give rise to NSs derived from the stroke-injured cortex. We demonstrate that these cells can be isolated from the cortex in two different models of stroke and from different stroke-lesioned cortical regions. Interestingly, SVZ NSCs give rise to a subpopulation of RAs in the cortex that contribute to astrogliosis and scar formation. Last, we show that these SVZ derived RAs can be converted to neurons in vivo by forced expression of Ascl1. Identifying the contribution of cells originating from the SVZ to injury repair has implications for neural regeneration strategies.
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Astrócitos/citologia , Córtex Cerebral/citologia , Ventrículos Laterais/citologia , Células-Tronco Neurais/citologia , Acidente Vascular Cerebral/patologia , Animais , Astrócitos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/biossíntese , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células-Tronco Neurais/metabolismo , Neurônios/metabolismo , Neurônios/patologia , Acidente Vascular Cerebral/metabolismoRESUMO
Alexander disease (AxD) is a neurodegenerative disorder with prominent white matter degeneration and the presence of Rosenthal fibers containing aggregates of glial fibrillary acidic protein (GFAP), and small stress proteins HSP27 and αB-crystallin, and widespread reactive gliosis. AxD is caused by mutations in GFAP, the main astrocyte intermediate filament protein. We previously showed that intermediate filament protein synemin is upregulated in reactive astrocytes after neurotrauma. Here, we examined immunohistochemically the presence of synemin in reactive astrocytes and Rosenthal fibers in two patients with AxD. There was an abundance of GFAP-positive Rosenthal fibers and widespread reactive gliosis in the white matter and subpial regions. Many of the GFAP-positive reactive astrocytes were positive for synemin, and synemin was also present in Rosenthal fibers. We show that synemin is expressed in reactive astrocytes in AxD, and is also present in Rosenthal fibers. The potential role of synemin in AxD pathogenesis remains to be investigated.
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Doença de Alexander/metabolismo , Astrócitos/metabolismo , Proteína Glial Fibrilar Ácida/metabolismo , Proteínas de Filamentos Intermediários/metabolismo , Adolescente , Doença de Alexander/genética , Doença de Alexander/patologia , Astrócitos/patologia , Encéfalo/metabolismo , Encéfalo/patologia , Criança , Feminino , Proteína Glial Fibrilar Ácida/genética , Gliose/metabolismo , Gliose/patologia , Humanos , Imuno-Histoquímica , Mutação Puntual , Distribuição TecidualRESUMO
Advances in cellular reprograming have shown that the delivery of specific transcription factors can result in the shift of one cell type to another. Brief forced expression of the four Yamanaka reprogramming factors (Klf4, Sox2, c-Myc, and Oct4) is able to convert many cell types into induced pluripotent stem cells, whereas some lineage specific transcription factors can convert cells from one type directly to another. Numerous strategies have already been developed for deriving neural cell types, with the hopes of better understanding/alleviating neurodegenerative disease. These cells facilitate drug discovery and constitute an autologous source of cells for brain repair, thus, avoiding rejection issues faced by allografts derived from embryonic stem cells. However, proper characterization of the various types of reprogrammed cells and an understanding of how these cells acquire neural fate is necessary before their translation into the clinic. Here, we review the progress, problems, and prospects with reprogrammed cell types with regards to neurodegenerative disease.
RESUMO
BACKGROUND: Astroglial cells are activated following injury and up-regulate the expression of the intermediate filament proteins glial fibrillary acidic protein (GFAP) and vimentin. Adult mice lacking the intermediate filament proteins GFAP and vimentin (GFAP(-/-)Vim(-/-)) show attenuated reactive gliosis, reduced glial scar formation and improved regeneration of neuronal synapses after neurotrauma. GFAP(-/-)Vim(-/-) mice exhibit larger brain infarcts after middle cerebral artery occlusion suggesting protective role of reactive gliosis after adult focal brain ischemia. However, the role of astrocyte activation and reactive gliosis in the injured developing brain is unknown. METHODOLOGY/PRINCIPAL FINDINGS: We subjected GFAP(-/-)Vim(-/-) and wild-type mice to unilateral hypoxia-ischemia (HI) at postnatal day 9 (P9). Bromodeoxyuridine (BrdU; 25 mg/kg) was injected intraperitoneally twice daily from P9 to P12. On P12 and P31, the animals were perfused intracardially. Immunohistochemistry with MAP-2, BrdU, NeuN, and S100 antibodies was performed on coronal sections. We found no difference in the hemisphere or infarct volume between GFAP(-/-)Vim(-/-) and wild-type mice at P12 and P31, i.e. 3 and 22 days after HI. At P31, the number of NeuN(+) neurons in the ischemic and contralateral hemisphere was comparable between GFAP(-/-)Vim(-/-) and wild-type mice. In wild-type mice, the number of S100(+) astrocytes was lower in the ipsilateral compared to contralateral hemisphere (65.0+/-50.1 vs. 85.6+/-34.0, p<0.05). In the GFAP(-/-)Vim(-/-) mice, the number of S100(+) astrocytes did not differ between the ischemic and contralateral hemisphere at P31. At P31, GFAP(-/-)Vim(-/-) mice showed an increase in NeuN(+)BrdU(+) (surviving newly born) neurons in the ischemic cortex compared to wild-type mice (6.7+/-7.7; n = 29 versus 2.9+/-3.6; n = 28, respectively, p<0.05), but a comparable number of S100(+)BrdU(+) (surviving newly born) astrocytes. CONCLUSIONS/SIGNIFICANCE: Our results suggest that attenuation of reactive gliosis in the developing brain does not affect the hemisphere or infarct volume after HI, but increases the number of surviving newborn neurons.