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1.
Adv Neurobiol ; 29: 117-162, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36255674

RESUMO

Proteoglycans, and especially their GAG components, participate in numerous biologically significant interactions with growth factors, chemokines, morphogens, guidance molecules, survival factors, and other extracellular and cell-surface components. These interactions are often critical to the basic developmental processes of cellular proliferation and differentiation, as well as to both the onset of disease sequelae and prevention of disease progression. In many tissues, proteoglycans and especially their glycosaminoglycan (GAG) components are mediators of these processes. The GAG family is characterized by covalently linked repeating disaccharides forming long unbranched polysaccharide chains. Thus far in higher eukaryotes, the family consists of chondroitin sulfate (CS), heparin/heparan sulfate (HS), dermatan sulfate (DS), keratan sulfate (KS) and hyaluronan (HA). All GAG chains (except HA) are characteristically modified by varying amounts of esterified sulfate. One or more GAG chains are usually found in nature bound to polypeptide backbones in the form of proteoglycans; HA is the exception. In the nervous system, GAG/proteoglycan-mediated interactions participate in proliferation and synaptogenesis, neural plasticity, and regeneration. This review focuses on the structure, chemistry and function of GAGs in nervous system development, disease, function and injury response.


Assuntos
Sulfatos de Condroitina , Glicosaminoglicanos , Humanos , Glicosaminoglicanos/metabolismo , Sulfatos de Condroitina/química , Sulfatos de Condroitina/metabolismo , Dermatan Sulfato , Sulfato de Queratano , Ácido Hialurônico , Heparitina Sulfato/metabolismo , Proteoglicanas , Heparina , Dissacarídeos , Sulfatos/metabolismo , Sistema Nervoso
2.
Front Cell Dev Biol ; 10: 745372, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35465334

RESUMO

The extracellular matrix (ECM) is critically important for most cellular processes including differentiation, morphogenesis, growth, survival and regeneration. The interplay between cells and the ECM often involves bidirectional signaling between ECM components and small molecules, i.e., growth factors, morphogens, hormones, etc., that regulate critical life processes. The ECM provides biochemical and contextual information by binding, storing, and releasing the bioactive signaling molecules, and/or mechanical information that signals from the cell membrane integrins through the cytoskeleton to the nucleus, thereby influencing cell phenotypes. Using these dynamic, reciprocal processes, cells can also remodel and reshape the ECM by degrading and re-assembling it, thereby sculpting their environments. In this review, we summarize the role of chondroitin sulfate proteoglycans as regulators of cell and tissue development using the skeletal growth plate model, with an emphasis on use of naturally occurring, or created mutants to decipher the role of proteoglycan components in signaling paradigms.

3.
J Neuroinflammation ; 18(1): 262, 2021 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-34749772

RESUMO

BACKGROUND: Neuronal ceroid lipofuscinoses, (NCLs or Batten disease) are a group of inherited, early onset, fatal neurodegenerative diseases associated with mutations in 13 genes. All forms of the disease are characterized by lysosomal accumulation of fluorescent storage material, as well as profound neurodegeneration, but the relationship of the various genes' function to a single biological process is not obvious. In this study, we used a well-characterized mouse model of classical late infantile NCL (cLINCL) in which the tripeptidyl peptidase 1 (Tpp1) gene is disrupted by gene targeting, resulting in loss of detectable TPP1 activity and leading to progressive neurological phenotypes including ataxia, increased motor deficiency, and early death. METHODS: In order to identify genes and pathways that may contribute to progression of the neurodegenerative process, we analyzed forebrain/midbrain and cerebellar transcriptional differences at 1, 2, 3 and 4 months of age in control and TPP1-deficient mice by global RNA-sequencing. RESULTS: Progressive neurodegenerative inflammatory responses involving microglia, astrocytes and endothelial cells were observed, accompanied by activation of leukocyte extravasation signals and upregulation of nitric oxide production and reactive oxygen species. Several astrocytic (i.e., Gfap, C4b, Osmr, Serpina3n) and microglial (i.e., Ctss, Itgb2, Itgax, Lyz2) genes were identified as strong markers for assessing disease progression as they showed increased levels of expression in vivo over time. Furthermore, transient increased expression of choroid plexus genes was observed at 2 months in the lateral and fourth ventricle, highlighting an early role for the choroid plexus and cerebrospinal fluid in the disease pathology. Based on these gene expression changes, we concluded that neuroinflammation starts, for the most part, after 2 months in the Tpp1-/- brain and that activation of microglia and astrocytes occur more rapidly in cerebellum than in the rest of the brain; confirming increased severity of inflammation in this region. CONCLUSIONS: These findings have led to a better understanding of cLINCL pathological onset and progression, which may aid in development of future therapeutic treatments for this disease.


Assuntos
Encéfalo/patologia , Lipofuscinoses Ceroides Neuronais/patologia , Transcriptoma , Animais , Modelos Animais de Doenças , Progressão da Doença , Camundongos , Camundongos Knockout , Lipofuscinoses Ceroides Neuronais/genética , Tripeptidil-Peptidase 1/genética
4.
Mol Ther ; 29(5): 1883-1902, 2021 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-33508430

RESUMO

Neonatal AAV9-gene therapy of the lysosomal enzyme galactosylceramidase (GALC) significantly ameliorates central and peripheral neuropathology, prolongs survival, and largely normalizes motor deficits in Twitcher mice. Despite these therapeutic milestones, new observations identified the presence of multiple small focal demyelinating areas in the brain after 6-8 months. These lesions are in stark contrast to the diffuse, global demyelination that affects the brain of naive Twitcher mice. Late-onset lesions exhibited lysosomal alterations with reduced expression of GALC and increased psychosine levels. Furthermore, we found that lesions were closely associated with the extravasation of plasma fibrinogen and activation of the fibrinogen-BMP-SMAD-GFAP gliotic response. Extravasation of fibrinogen correlated with tight junction disruptions of the vasculature within the lesioned areas. The lesions were surrounded by normal appearing white matter. Our study shows that the dysregulation of therapeutic GALC was likely driven by the exhaustion of therapeutic AAV episomal DNA within the lesions, paralleling the presence of proliferating oligodendrocyte progenitors and glia. We believe that this is the first demonstration of diminishing expression in vivo from an AAV gene therapy vector with detrimental effects in the brain of a lysosomal storage disease animal model. The development of this phenotype linking localized loss of GALC activity with relapsing neuropathology in the adult brain of neonatally AAV-gene therapy-treated Twitcher mice identifies and alerts to possible late-onset reductions of AAV efficacy, with implications to other genetic leukodystrophies.


Assuntos
Galactosilceramidase/genética , Terapia Genética/métodos , Leucodistrofia de Células Globoides/patologia , Substância Branca/patologia , Animais , Animais Recém-Nascidos , Células Cultivadas , Dependovirus/genética , Modelos Animais de Doenças , Feminino , Fibrinogênio/metabolismo , Galactosilceramidase/metabolismo , Vetores Genéticos/administração & dosagem , Leucodistrofia de Células Globoides/sangue , Leucodistrofia de Células Globoides/genética , Leucodistrofia de Células Globoides/terapia , Masculino , Camundongos , Recidiva
5.
Elife ; 92020 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-32909945

RESUMO

The umbilical artery lumen closes rapidly at birth, preventing neonatal blood loss, whereas the umbilical vein remains patent longer. Here, analysis of umbilical cords from humans and other mammals identified differential arterial-venous proteoglycan dynamics as a determinant of these contrasting vascular responses. The umbilical artery, but not the vein, has an inner layer enriched in the hydrated proteoglycan aggrecan, external to which lie contraction-primed smooth muscle cells (SMC). At birth, SMC contraction drives inner layer buckling and centripetal displacement to occlude the arterial lumen, a mechanism revealed by biomechanical observations and confirmed by computational analyses. This vascular dimorphism arises from spatially regulated proteoglycan expression and breakdown. Mice lacking aggrecan or the metalloprotease ADAMTS1, which degrades proteoglycans, demonstrate their opposing roles in umbilical vascular dimorphism, including effects on SMC differentiation. Umbilical vessel dimorphism is conserved in mammals, suggesting that differential proteoglycan dynamics and inner layer buckling were positively selected during evolution.


Assuntos
Agrecanas/metabolismo , Miócitos de Músculo Liso , Artérias Umbilicais , Proteína ADAMTS1/metabolismo , Animais , Diferenciação Celular/fisiologia , Feminino , Humanos , Camundongos Transgênicos , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/fisiologia , Parto/fisiologia , Gravidez , Artérias Umbilicais/citologia , Artérias Umbilicais/metabolismo , Artérias Umbilicais/fisiologia
6.
JBMR Plus ; 4(2): e10254, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32083237

RESUMO

Osteoarthritis and osteoporosis are widely prevalent and have far-reaching public health implications. There is increasing evidence that epigenetics, in particular, histone 3 lysine 79 methyltransferase DOT1L, plays an important role in the cartilage and bone biology. In this study, we evaluated the role of Dot1l in the articular cartilage, growth plate, and trabecular bone utilizing conditional KO mouse models. We generated chondrocyte-specific constitutive and inducible conditional Dot1l KO mouse lines using Col2a1-Cre and Acan-CreER systems. Prenatal deletion of Dot1l in mouse chondrocytes led to perinatal mortality, accelerated ossification, and dysregulation of Col10a1 expression. Postnatal deletion of Dot1l in mouse chondrocytes resulted in trabecular bone loss decreased extracellular matrix production, and disruption of the growth plate. In addition, pharmacological inhibition of DOT1L in a progeria mouse model partially rescued the abnormal osseous phenotype. In conclusion, Dot1l is important in maintaining the growth plate, extracellular matrix production, and trabecular bone. © 2019 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

7.
ASN Neuro ; 11: 1759091419843393, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31003587

RESUMO

In humans, homozygous mutations in the TPP1 gene results in loss of tripeptidyl peptidase 1 (TPP1) enzymatic activity, leading to late infantile neuronal ceroid lipofuscinoses disease. Using a mouse model that targets the Tpp1 gene and recapitulates the pathology and clinical features of the human disease, we analyzed end-stage (4 months) transcriptional changes associated with lack of TPP1 activity. Using RNA sequencing technology, Tpp1 expression changes in the forebrain/midbrain and cerebellum of 4-month-old homozygotes were compared with strain-related controls. Transcriptional changes were found in 510 and 1,550 gene transcripts in forebrain/midbrain and cerebellum, respectively, from Tpp1-deficient brain tissues when compared with age-matched controls. Analysis of the differentially expressed genes using the Ingenuity™ pathway software, revealed increased neuroinflammation activity in microglia and astrocytes that could lead to neuronal dysfunction, particularly in the cerebellum. We also observed upregulation in the production of nitric oxide and reactive oxygen species; activation of leukocyte extravasation signals and complement pathways; and downregulation of major transcription factors involved in control of circadian rhythm. Several of these expression changes were confirmed by independent quantitative polymerase chain reaction and histological analysis by mRNA in situ hybridization, which allowed for an in-depth anatomical analysis of the pathology and provided independent confirmation of at least two of the major networks affected in this model. The identification of differentially expressed genes has revealed new lines of investigation for this complex disorder that may lead to novel therapeutic targets.


Assuntos
Aminopeptidases/genética , Dipeptidil Peptidases e Tripeptidil Peptidases/genética , Regulação da Expressão Gênica/fisiologia , Lipofuscinoses Ceroides Neuronais/genética , Lipofuscinoses Ceroides Neuronais/metabolismo , Serina Proteases/genética , Transcriptoma/fisiologia , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Modelos Animais de Doenças , Camundongos , Mutação , Lipofuscinoses Ceroides Neuronais/patologia , Tripeptidil-Peptidase 1
8.
FEBS Lett ; 592(23): 3791-3805, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-29513405

RESUMO

Proteoglycans are diverse, complex extracellular/cell surface macromolecules composed of a central core protein with covalently linked glycosaminoglycan (GAG) chains; both of these components contribute to the growing list of important bio-active functions attributed to proteoglycans. Increasingly, attention has been paid to the roles of proteoglycans in nervous tissue development due to their highly regulated spatio/temporal expression patterns, whereby they promote/inhibit neurite outgrowth, participate in specification and maturation of various precursor cell types, and regulate cell behaviors like migration, axonal pathfinding, synaptogenesis and plasticity. These functions emanate from both the environments proteoglycans create around cells by retaining ions and water or serving as scaffolds for cell shaping or motility, and from dynamic interactions that modulate signaling fields for cytokines, growth factors and morphogens, which may bind to either the protein or GAG portions. Also, genetic abnormalities impacting proteoglycan synthesis during critical steps of brain development and response to environmental insults and injuries, as well as changes in microenvironment interactions leading to tumors in the central nervous system, all suggest roles for proteoglycans in behavioral and intellectual disorders and malignancies.


Assuntos
Neoplasias Encefálicas/metabolismo , Encéfalo/metabolismo , Sistema Nervoso Central/metabolismo , Proteoglicanas/metabolismo , Animais , Encéfalo/citologia , Neoplasias Encefálicas/genética , Movimento Celular/genética , Plasticidade Celular/genética , Sistema Nervoso Central/citologia , Perfilação da Expressão Gênica , Humanos , Neurogênese/genética , Proteoglicanas/química
9.
ACS Chem Neurosci ; 6(3): 494-504, 2015 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-25688887

RESUMO

Luminescent semiconductor ∼9.5 nm nanoparticles (quantum dots: QDs) have intrinsic physiochemical and optical properties which enable us to begin to understand the mechanisms of nanoparticle mediated chemical/drug delivery. Here, we demonstrate the ability of CdSe/ZnS core/shell QDs surface functionalized with a zwitterionic compact ligand to deliver a cell-penetrating lipopeptide to the developing chick embryo brain without any apparent toxicity. Functionalized QDs were conjugated to the palmitoylated peptide WGDap(Palmitoyl)VKIKKP9GGH6, previously shown to uniquely facilitate endosomal escape, and microinjected into the embryonic chick spinal cord canal at embryo day 4 (E4). We were subsequently able to follow the labeling of spinal cord extension into the ventricles, migratory neuroblasts, maturing brain cells, and complex structures such as the choroid plexus. QD intensity extended throughout the brain, and peaked between E8 and E11 when fluorescence was concentrated in the choroid plexus before declining to hatching (E21/P0). We observed no abnormalities in embryonic patterning or embryo survival, and mRNA in situ hybridization confirmed that, at key developmental stages, the expression pattern of genes associated with different brain cell types (brain lipid binding protein, Sox-2, proteolipid protein and Class III-ß-Tubulin) all showed a normal labeling pattern and intensity. Our findings suggest that we can use chemically modified QDs to identify and track neural stem cells as they migrate, that the choroid plexus clears these injected QDs/nanoparticles from the brain after E15, and that they can deliver drugs and peptides to the developing brain.


Assuntos
Encéfalo , Peptídeos/metabolismo , Pontos Quânticos/metabolismo , Ácido 8,11,14-Eicosatrienoico/metabolismo , Animais , Animais Recém-Nascidos , Encéfalo/efeitos dos fármacos , Encéfalo/embriologia , Encéfalo/metabolismo , Embrião de Galinha , Sistemas de Liberação de Medicamentos , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Microinjeções , Microscopia de Fluorescência , Peptídeos/química , Peptídeos/genética , Pontos Quânticos/química , RNA Mensageiro , Medula Espinal/efeitos dos fármacos , Medula Espinal/embriologia , Medula Espinal/metabolismo
10.
J Struct Biol ; 190(1): 56-72, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25682762

RESUMO

Myelin - the multilayer membrane that envelops axons - is a facilitator of rapid nerve conduction. Oligodendrocytes form CNS myelin; the prevailing hypothesis being that they do it by extending a process that circumnavigates the axon. It is pertinent to ask how myelin is built because oligodendrocyte plasma membrane and myelin are compositionally different. To this end, we examined oligodendrocyte cultures and embryonic avian optic nerves by electron microscopy, immuno-electron microscopy and three-dimensional electron tomography. The results support three novel concepts. Myelin membranes are synthesized as tubules and packaged into "myelinophore organelles" in the oligodendrocyte perikaryon. Myelin membranes are matured in and transported by myelinophore organelles within an oligodendrocyte process. The myelin sheath is generated by myelin membrane fusion inside an oligodendrocyte process. These findings abrogate the dogma of myelin resulting from a wrapping motion of an oligodendrocyte process and open up new avenues in the quest for understanding myelination in health and disease.


Assuntos
Bainha de Mielina/ultraestrutura , Oligodendroglia/ultraestrutura , Animais , Axônios/fisiologia , Axônios/ultraestrutura , Membrana Celular/fisiologia , Membrana Celular/ultraestrutura , Células Cultivadas , Sistema Nervoso Central/citologia , Embrião de Galinha , Bainha de Mielina/fisiologia , Oligodendroglia/fisiologia , Organelas/ultraestrutura , Carneiro Doméstico , Processos Estocásticos
11.
Dev Biol ; 396(2): 224-36, 2014 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-25446537

RESUMO

The proteoglycan aggrecan is a prominent component of the extracellular matrix in growth plate cartilage. A naturally occurring, recessive, perinatally lethal mutation in the aggrecan core protein gene, cmd(bc) (Acan(cmd-Bc)), that deletes the entire protein-coding sequence provided a model in which to characterize the phenotypic and morphologic effects of aggrecan deletion on skeletal development. We also generated a novel transgenic mouse, Tg(COL2A1-ACAN), that has the chick ACAN coding sequence driven by the mouse COL2A1 promoter to enable the production of cmd(bc)/cmd(bc); Tg(COL2A1-ACAN) rescue embryos. These were used to assess the impact of aggrecan on growth plate organization, chondrocyte survival and proliferation, and the expression of mRNAs encoding chondrocyte differentiation markers and growth factors. Homozygous mutant (cmd(bc)/cmd(bc)) embryos exhibited severe defects in all skeletal elements with deformed and shortened (50%) limb elements. Expression of aggrecan in rescue embryos reversed the skeletal defects to varying degrees with a 20% increase in limb element length and near-full reversal (80%) of size and diameter of the ribcage and vertebrae. Aggrecan-null growth plates were devoid of matrix and lacked chondrocyte organization and differentiation, while those of the rescue embryos exhibited matrix production concomitant with partial zonation of chondrocytes having proliferative and hypertrophic morphologies. Deformation of the trachea, likely the cause of the mutation's lethality, was reduced in the rescue embryos. Aggrecan-null embryos also had abnormal patterns of COL10A1, SOX9, IHH, PTCH1, and FGFR3 mRNA expression in the growth plate. Expression of chick aggrecan in the rescue embryos notably increased COLX expression, accompanied by the reappearance of a hypertrophic zone and IHH expression. Significantly, in transgenic rescue embryos, the cell death and decreased proliferation phenotypes exhibited by the mutants were reversed; both were restored to wild-type levels. These findings suggest that aggrecan has a major role in regulating the expression of key growth factors and signaling molecules during development of cartilaginous tissue and is essential for proper chondrocyte organization, morphology, and survival during embryonic limb development.


Assuntos
Agrecanas/genética , Agrecanas/metabolismo , Diferenciação Celular/fisiologia , Condrócitos/fisiologia , Extremidades/embriologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Lâmina de Crescimento/embriologia , Agrecanas/deficiência , Animais , Southern Blotting , Proliferação de Células , Galinhas , Condrócitos/metabolismo , Primers do DNA/genética , Lâmina de Crescimento/citologia , Proteínas Hedgehog/metabolismo , Imuno-Histoquímica , Hibridização In Situ , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos Transgênicos , Fatores de Transcrição SOX9/metabolismo
12.
Adv Neurobiol ; 9: 89-115, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25151376

RESUMO

The glycosaminoglycan (GAG) family is characterized by covalently linked repeating disaccharides forming long unbranched polysaccharide chains. Thus far in higher eukaryotes, the family consists of chondroitin sulfate (CS), heparin/heparan sulfate (HS), dermatan sulfate (DS), and hyaluronan (HA). All GAG chains (except HA) are characteristically modified by varying amounts of esterified sulfate. One or more GAG chains are usually found in nature bound to polypeptide backbones in the form of proteoglycans; HA is the exception and is not synthesized covalently bound to a protein. Proteoglycans, and especially their GAG components, participate in numerous biologically significant interactions with growth factors, chemokines, morphogens, guidance molecules, survival factors, and other extracellular and cell-surface components. These interactions are often critical to the basic developmental processes of cellular proliferation and differentiation, as well as to both the onset of disease sequelae and the prevention of disease progression. In the nervous system, GAG/proteoglycan-mediated interactions participate in proliferation and synaptogenesis, neural plasticity, and regeneration. This review focuses on the structure, chemistry, and function of GAGs in nervous system development, disease, and injury response.

13.
Eur J Neurosci ; 34(12): 1906-22, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22132705

RESUMO

Oligodendrocytes--best known for assembling central nervous system myelin--can be categorized as precursors, myelin-forming cells and non-myelinating perineuronal cells. Perineuronal oligodendrocytes have been well characterized morphologically and ultrastructurally, but knowledge about their function remains scanty. It has been proposed that perineuronal oligodendrocytes support neurons and, following injury, transform into myelin-synthesizing cells. Recent findings implicating perineuronal oligodendrocytes in cytoarchitectural abnormalities in the prefrontal cortex of schizophrenia and other psychiatric disorders shed new light on these cells. We have obtained the genetic signature of perineuronal oligodendrocytes by identifying gene expression differences between oligodendrocyte subpopulations using cell-specific tags, microarray technology, quantitative time-resolved polymerase chain reaction and bioinformatics tools. We show that perineuronal cells are the progeny of oligodendrocyte progenitors and, hence, are members of the oligodendrocyte lineage. Physiologically they exhibit a novel phenotype. Their expression of PDGFR-αß and its growth factor ligand PDGF-CC sets them apart from members of their lineage as this receptor precludes their response to the same growth factors that act on myelinating cells. Their coordinate expression and context-specific usage of transcription factors Olig2, Ascl1 and Pax6, together with the prominent presence of transcription factors Pea3, Lhx2 and Otx2--not hitherto linked to the oligodendrocyte lineage--suggested a cell with features that blur the boundary between a neuron and a glial cell. But they also maintain a reservoir of untranslated transcripts encoding major myelin proteins presumably for a demyelinating episode. This first molecular characterization of perineuronal oligodendrocytes revealed the striking difference between the myelinating and non-myelinating phenotypes.


Assuntos
Linhagem da Célula , Expressão Gênica , Oligodendroglia/fisiologia , Fenótipo , Animais , Biomarcadores/metabolismo , Células Cultivadas , Perfilação da Expressão Gênica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Análise em Microsséries , Proteínas da Mielina/genética , Proteínas da Mielina/metabolismo , Bainha de Mielina/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Oligodendroglia/citologia , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
14.
Brain Res ; 1389: 35-49, 2011 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-21396923

RESUMO

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.


Assuntos
Astrócitos/citologia , Lesões Encefálicas/patologia , Células-Tronco/citologia , Animais , Apoptose/fisiologia , Astrócitos/metabolismo , Northern Blotting , Lesões Encefálicas/metabolismo , Diferenciação Celular , Movimento Celular/fisiologia , Proliferação de Células , Embrião de Galinha , Embrião não Mamífero , Feminino , Imuno-Histoquímica , Hibridização In Situ , Inflamação/metabolismo , Inflamação/patologia , Modelos Animais , Células-Tronco/metabolismo
15.
J Neurochem ; 117(2): 187-96, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21070241

RESUMO

Cold pre-conditioning reduces subsequent brain injury in small animals but the underlying mechanisms remain undefined. As hypothermia triggers systemic macrophage tumor necrosis factor alpha (TNF-α) production and other neural pre-conditioning stimuli depend on this cytokine, we reasoned that microglia and TNF-α would be similarly involved with cold pre-conditioning neuroprotection. Also, as slice cultures closely approximate their in vivo counterpart and include quiescent microglia, we used rat hippocampal slice cultures to confirm this hypothesis. Furthermore, inflammatory cytokine gene screening with subsequent PCR and immunostaining confirmation of targeted mRNA and related protein changes showed that cold pre-conditioning triggered a significant rise in TNF-α that localized to microglia and a significant rise in interleukin (IL)-11 that localized mainly to hippocampal pyramidal neurons and, more rarely, astrocytes. Importantly, co-stimulation with cold and IL-11, an anti-inflammatory cytokine that inhibits TNF-α expression, abrogated the otherwise evident protection. Instead, cold pre-conditioning coupled with blockade of IL-11 signaling further enhanced neuroprotection from that seen with cold pre-conditioning alone. Thus, physiological activation of brain pro-inflammatory cytokine signaling, and its amplification by inhibition of coincident anti-inflammatory cytokine signaling, may be opportune targets for the development of novel therapeutics that can mimic the protection seen in cold pre-conditioning.


Assuntos
Temperatura Baixa , Interleucina-11/antagonistas & inibidores , Interleucina-11/metabolismo , Neurônios/fisiologia , Fator de Necrose Tumoral alfa/metabolismo , Análise de Variância , Animais , Animais Recém-Nascidos , Anticorpos/farmacologia , Região CA1 Hipocampal/citologia , Meios de Cultura Livres de Soro/farmacologia , Citocinas/genética , Citocinas/imunologia , Citocinas/metabolismo , Agonistas de Aminoácidos Excitatórios/toxicidade , Regulação da Expressão Gênica/efeitos dos fármacos , Proteína Glial Fibrilar Ácida/metabolismo , N-Metilaspartato/toxicidade , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Fosfopiruvato Hidratase/metabolismo , RNA Mensageiro/metabolismo , Ratos , Receptores Tipo I de Fatores de Necrose Tumoral/farmacologia , Transdução de Sinais/efeitos dos fármacos , Temperatura , Fatores de Tempo , Técnicas de Cultura de Tecidos , Fator de Necrose Tumoral alfa/genética
16.
Dev Biol ; 329(2): 242-57, 2009 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-19268444

RESUMO

Chick and mouse embryos with heritable deficiencies of aggrecan exhibit severe dwarfism and premature death, demonstrating the essential involvement of aggrecan in development. The aggrecan-deficient nanomelic (nm) chick mutant E12 fully formed growth plate (GP) is devoid of matrix and exhibits markedly altered cytoarchitecture, proliferative capacity, and degree of cell death. While differentiation of chondroblasts to pre-hypertrophic chondrocytes (IHH expression) is normal up to E6, the extended periosteum expression pattern of PTCH (a downstream effector of IHH) indicates altered propagation of IHH signaling, as well as accelerated down-regulation of FGFR3 expression, decreased BrdU incorporation and higher levels of ERK phosphorylation, all indicating early effects on FGF signaling. By E7 reduced IHH expression and premature expression of COL10A1 foreshadow the acceleration of hypertrophy observed at E12. By E8, exacerbated co-expression of IHH and COL10A1 lead to delayed separation and establishment of the two GPs in each element. By E9, increased numbers of cells express P-SMAD1/5/8, indicating altered BMP signaling. These results indicate that the IHH, FGF and BMP signaling pathways are altered from the very beginning of GP formation in the absence of aggrecan, thereby inducing premature hypertrophic chondrocyte maturation, leading to the nanomelic long bone growth disorder.


Assuntos
Agrecanas/fisiologia , Lâmina de Crescimento/embriologia , Morfogênese , Animais , Sequência de Bases , Embrião de Galinha , Primers do DNA , Fatores de Crescimento de Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Imuno-Histoquímica , Hibridização In Situ , Camundongos , Fosforilação , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais
17.
J Neurosci Methods ; 177(1): 30-43, 2009 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-18948137

RESUMO

Compared to our knowledge of neurogenesis, relatively little is known about glial cell specification and migration during central nervous system development. We have established a novel chick hindbrain slice preparation which permits examination of gliogenesis in its native environment, providing a means to study the signaling pathways involved in glial cell specification and migration during development. Cells in the hindbrain slice preparations mature in a manner which is similar to in vivo developmental timing and patterning paradigms. To demonstrate the utility of this approach, we examined the effect of the retinoic acid signaling pathway on cells in these slices, showing that addition of exogenous trans-retinoic acid to slice cultures promotes expression of a marker of mature astrocytes, glial fibrillary acidic protein (GFAP), while the inhibition of endogenous retinoic acid synthesis reduces GFAP expression; the results suggest a role for retinoic acid in modulating glial differentiation. Using these hindbrain slice cultures, we have used two different approaches to label glial progenitors specifically at the ventricular zone and have observed for the first time the ventrally-directed migration of these cells from the ventricular zone of the hindbrain. This slice culture system is thus an innovative and robust tool for examining glial cell migration and the extracellular molecular and signaling pathways which regulate glial differentiation.


Assuntos
Movimento Celular/fisiologia , Neuroglia/fisiologia , Rombencéfalo/citologia , Rombencéfalo/embriologia , Fatores Etários , Animais , Antineoplásicos/farmacologia , Bromodesoxiuridina/metabolismo , Caspase 3/metabolismo , Caspase 7/metabolismo , Diferenciação Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Embrião de Galinha , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteína Glial Fibrilar Ácida/genética , Proteína Glial Fibrilar Ácida/metabolismo , Proteínas de Filamentos Intermediários/genética , Proteínas de Filamentos Intermediários/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Nestina , Neuroglia/efeitos dos fármacos , Técnicas de Cultura de Órgãos , Fosfopiruvato Hidratase/genética , Fosfopiruvato Hidratase/metabolismo , Timidina/metabolismo , Fatores de Tempo , Tretinoína/farmacologia , Proteínas Supressoras de Tumor/metabolismo , p-Aminoazobenzeno/análogos & derivados , p-Aminoazobenzeno/farmacologia
18.
Dev Biol ; 315(1): 114-24, 2008 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-18207138

RESUMO

Determination of the molecules that regulate astrocyte development has been hindered by the paucity of markers that identify astrocytic precursors in vivo. Here we report that the chondroitin sulfate proteoglycan aggrecan both regulates astrocyte development and is expressed by embryonic glial precursors. During chick brain development, the onset of aggrecan expression precedes that of the astrocytic marker GFAP and is concomitant with detection of the early glial markers GLAST and glutamine synthetase. In co-expression studies, we established that aggrecan-rich cells contain the radial glial markers nestin, BLBP and GLAST and later in embryogenesis, the astroglial marker GFAP. Parallel in vitro studies showed that ventricular zone cultures, enriched in aggrecan-expressing cells, could be directed to a GFAP-positive fate in G5-supplemented differentiation media. Analysis of the chick aggrecan mutant nanomelia revealed marked increases in the expression of the astrocyte differentiation genes GFAP, GLAST and GS in the absence of extracellular aggrecan. These increases in astrocytic marker gene expression could not be accounted for by changes in precursor proliferation or cell death, suggesting that aggrecan regulates the rate of astrocyte differentiation. Taken together, these results indicate a major role for aggrecan in the control of glial cell maturation during brain development.


Assuntos
Agrecanas/metabolismo , Astrócitos/fisiologia , Encéfalo/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Neuroglia/metabolismo , Agrecanas/genética , Animais , Biomarcadores , Encéfalo/citologia , Técnicas de Cultura de Células , Diferenciação Celular , Linhagem da Célula , Células Cultivadas , Embrião de Galinha , DNA Complementar , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Proteína Glial Fibrilar Ácida/genética , Proteína Glial Fibrilar Ácida/metabolismo , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Cinética , Mesencéfalo/citologia , Mutação , RNA Mensageiro/análise
19.
J Biol Chem ; 280(42): 35606-16, 2005 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-16100116

RESUMO

Expression of the extracellular proteoglycan aggrecan is both cell-specific and developmentally regulated. Previous studies identified six functionally defined cis elements in the aggrecan promoter region which were shown to repress aggrecan gene expression (1). Using competition electrophoretic mobility shift assays (EMSAs) we have now identified in nuclear extracts a functional repressor cis element, (T/C)TCCCCT(A/C)RRC, which occurs at multiple locations within the chick aggrecan regulatory region. We purified the factor that binds to this cis element and established that it, APBP-1 (aggrecan promoter-binding protein-1), is a 19-kDa protein that has significant homology to CIRP (cold inducible RNA-binding protein). Recombinantly expressed APBP-1 mimics the native cis element-trans factor interaction in EMSAs. In situ hybridization demonstrates that aggrecan and APBP-1 RNA expression are restricted to complementary tissues in the developing limb, and Northern blot analysis of chick limb bud mRNA shows that APBP-1 mRNA expression is inversely correlated with aggrecan mRNA expression. Functional analyses by transient transfections and Northern blot analyses suggest APBP-1 has the capacity to repress aggrecan expression, indicating that this factor may be important regulator of aggrecan gene expression.


Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/fisiologia , Proteínas da Matriz Extracelular/química , Regulação da Expressão Gênica , Lectinas Tipo C/química , Proteoglicanas/química , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/fisiologia , Agrecanas , Sequência de Aminoácidos , Animais , Ligação Competitiva , Northern Blotting , Southern Blotting , Western Blotting , Galinhas , Cromatografia Líquida de Alta Pressão , Clonagem Molecular , DNA/química , Eletroforese em Gel de Poliacrilamida , Hibridização In Situ , Espectrometria de Massas , Dados de Sequência Molecular , Oligonucleotídeos/química , Regiões Promotoras Genéticas , Ligação Proteica , Estrutura Terciária de Proteína , RNA/química , RNA Mensageiro/metabolismo , Transcrição Gênica , Transfecção
20.
Biophys Chem ; 108(1-3): 245-58, 2004 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-15043933

RESUMO

The nervous system is generated from cells lining the ventricular system. Our understanding of the fate potentials and lineage relationships of these cells is being re-evaluated, both because of recent demonstrations that radial glia can generate neurons and because of the identification of fate-determining genes. A variety of intrinsic and extrinsic molecules, including proteoglycans, regulate embryonic and postnatal brain development. Using probes modeled after species conserved domains of heparan sulfate proteoglycans, we cloned a novel gene called novocan, raised monoclonal antibodies against a segment of the predicted amino acid sequence of the expressed protein (NOVOcan) and used the antibodies to establish the cell and tissue localization of NOVOcan in postnatal rat brains by immunohistochemistry. NOVOcan was expressed in cells lining the ventricles, including a variety of radial glia during early postnatal development. Later, as radial glia disappeared and ependymal cells appeared, NOVOcan was detected in ependymal cells and in tanycytes, a specialized form of ependymal cell resembling radial glia. NOVOcan was absent in two known progeny of radial glia, mature astrocytes and neurons. Whereas NOVOcan was also absent in mature oligodendrocytes (OLGs), it was present in OLG precursors in developing white matter. These studies set the stage for determining the roles of NOVOcan in brain cell lineage patterns as well as in other aspects of development.


Assuntos
Proteínas do Tecido Nervoso/metabolismo , Neuroglia/citologia , Neurônios/metabolismo , Adulto , Animais , Linhagem da Célula/fisiologia , Sistema Nervoso Central/citologia , Sistema Nervoso Central/embriologia , Sistema Nervoso Central/crescimento & desenvolvimento , Ventrículos Cerebrais/citologia , Ventrículos Cerebrais/metabolismo , Clonagem Molecular , Desenvolvimento Embrionário e Fetal/fisiologia , Epêndima/citologia , Epêndima/metabolismo , Regulação da Expressão Gênica , Humanos , Imuno-Histoquímica , Proteínas do Tecido Nervoso/análise , Proteínas do Tecido Nervoso/genética , Neuroglia/metabolismo , Neurônios/citologia , Proteoglicanas/metabolismo , RNA Mensageiro/biossíntese , Ratos
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