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1.
Mol Neurobiol ; 55(7): 5478-5489, 2018 Jul.
Article in English | MEDLINE | ID: mdl-28956310

ABSTRACT

Vimentin is an intermediate filament (also known as nanofilament) protein expressed in several cell types of the central nervous system, including astrocytes and neural stem/progenitor cells. Mutation of the vimentin serine sites that are phosphorylated during mitosis (VIM SA/SA ) leads to cytokinetic failures in fibroblasts and lens epithelial cells, resulting in chromosomal instability and increased expression of cell senescence markers. In this study, we investigated morphology, proliferative capacity, and motility of VIM SA/SA astrocytes, and their effect on the differentiation of neural stem/progenitor cells. VIM SA/SA astrocytes expressed less vimentin and more GFAP but showed a well-developed intermediate filament network, exhibited normal cell morphology, proliferation, and motility in an in vitro wound closing assay. Interestingly, we found a two- to fourfold increased neuronal differentiation of VIM SA/SA neurosphere cells, both in a standard 2D and in Bioactive3D cell culture systems, and determined that this effect was neurosphere cell autonomous and not dependent on cocultured astrocytes. Using BrdU in vivo labeling to assess neural stem/progenitor cell proliferation and differentiation in the hippocampus of adult mice, one of the two major adult neurogenic regions, we found a modest increase (by 8%) in the fraction of newly born and surviving neurons. Thus, mutation of the serine sites phosphorylated in vimentin during mitosis alters intermediate filament protein expression but has no effect on astrocyte morphology or proliferation, and leads to increased neuronal differentiation of neural progenitor cells.


Subject(s)
Cell Differentiation , Neural Stem Cells/cytology , Neurons/cytology , Vimentin/deficiency , Vimentin/metabolism , Animals , Astrocytes/cytology , Astrocytes/metabolism , Cell Proliferation , Cell Survival , Dentate Gyrus/cytology , Intermediate Filaments/metabolism , Mice, Inbred C57BL , Neurogenesis , Phosphorylation , Spheroids, Cellular/cytology , Wound Healing
2.
Cereb Cortex ; 27(12): 5672-5682, 2017 12 01.
Article in English | MEDLINE | ID: mdl-27979877

ABSTRACT

Adult neurogenesis in human brain is known to occur in the hippocampus, the subventricular zone, and the striatum. Neural progenitor cells (NPCs) were reported in the cortex of epilepsy patients; however, their identity is not known. Since astrocytes were proposed as the source of neural progenitors in both healthy and diseased brain, we tested the hypothesis that NPCs in the epileptic cortex originate from reactive, alternatively, de-differentiated astrocytes that express glutamate aspartate transporter (GLAST). We assessed the capacity to form neurospheres and the differentiation potential of cells dissociated from fresh cortical tissue from patients who underwent surgical treatment for pharmacologically intractable epilepsy. Neurospheres were generated from 57% of cases (8/14). Upon differentiation, the neurosphere cells gave rise to neurons, oligodendrocytes, and astrocytes. Sorting of dissociated cells showed that only cells negative for GLAST formed neurospheres. In conclusion, we show that cells with neural stem cell properties are present in brain cortex of epilepsy patients, and that these cells are not GLAST-positive astrocytes.


Subject(s)
Astrocytes/metabolism , Cerebral Cortex/metabolism , Drug Resistant Epilepsy/metabolism , Excitatory Amino Acid Transporter 1/metabolism , Neural Stem Cells/metabolism , Neurogenesis/physiology , Adolescent , Adult , Astrocytes/pathology , Cells, Cultured , Cerebral Cortex/pathology , Cerebral Cortex/surgery , Child , Child, Preschool , Drug Resistant Epilepsy/pathology , Drug Resistant Epilepsy/surgery , Female , Gray Matter/metabolism , Gray Matter/pathology , Gray Matter/surgery , Humans , Male , Middle Aged , Multipotent Stem Cells/metabolism , Multipotent Stem Cells/pathology , Neural Stem Cells/pathology , Young Adult
3.
Neurochem Res ; 40(2): 336-52, 2015 Feb.
Article in English | MEDLINE | ID: mdl-25249434

ABSTRACT

Brain tumors are heterogeneous with respect to genetic and histological properties of cells within the tumor tissue. To study subpopulations of cells, we developed a protocol for obtaining viable single cells from freshly isolated human brain tissue for single cell gene expression profiling. We evaluated this technique for characterization of cell populations within brain tumor and tumor penumbra. Fresh tumor tissue was obtained from one astrocytoma grade IV and one oligodendroglioma grade III tumor as well as the tumor penumbra of the latter tumor. The tissue was dissociated into individual cells and the expression of 36 genes was assessed by reverse transcription quantitative PCR followed by data analysis. We show that tumor cells from both the astrocytoma grade IV and oligodendroglioma grade III tumor constituted cell subpopulations defined by their gene expression profiles. Some cells from the oligodendroglioma grade III tumor proper shared molecular characteristics with the cells from the penumbra of the same tumor suggesting that a subpopulation of cells within the oligodendroglioma grade III tumor consisted of normal brain cells. We conclude that subpopulations of tumor cells can be identified by using single cell gene expression profiling.


Subject(s)
Brain Neoplasms/genetics , Brain Neoplasms/pathology , Gene Expression Profiling , Single-Cell Analysis , Astrocytoma/genetics , Astrocytoma/pathology , Brain Neoplasms/classification , Humans , Male , Middle Aged , Oligodendroglioma/genetics , Oligodendroglioma/pathology
4.
J Neurochem ; 128(6): 878-89, 2014 Mar.
Article in English | MEDLINE | ID: mdl-24188029

ABSTRACT

Heparin-binding epidermal growth factor-like growth factor (HB-EGF), a vascular-derived trophic factor, belongs to the epidermal growth factor (EGF) family of neuroprotective, hypoxia-inducible proteins released by astrocytes in CNS injuries. It was suggested that HB-EGF can replace fetal calf serum (FCS) in astrocyte cultures. We previously demonstrated that in contrast to standard 2D cell culture systems, Bioactive3D culture system, when used with FCS, minimizes the baseline activation of astrocytes and preserves their complex morphology. Here, we show that HB-EGF induced EGF receptor (EGFR) activation by Y1068 phosphorylation, Mapk/Erk pathway activation, and led to an increase in cell proliferation, more prominent in Bioactive3D than in 2D cultures. HB-EGF changed morphology of 2D and Bioactive3D cultured astrocytes toward a radial glia-like phenotype and induced the expression of intermediate filament and progenitor cell marker protein nestin. Glial fibrillary acidic protein (GFAP) and vimentin protein expression was unaffected. RT-qPCR analysis demonstrated that HB-EGF affected the expression of Notch signaling pathway genes, implying a role for the Notch signaling in HB-EGF-mediated astrocyte response. HB-EGF can be used as a FCS replacement for astrocyte expansion and in vitro experimentation both in 2D and Bioactive3D culture systems; however, caution should be exercised since it appears to induce partial de-differentiation of astrocytes.


Subject(s)
Astrocytes/cytology , Astrocytes/metabolism , Intercellular Signaling Peptides and Proteins/physiology , Intermediate Filament Proteins/metabolism , MAP Kinase Signaling System/physiology , Animals , Astrocytes/drug effects , Cell Culture Techniques/methods , Cell Differentiation/drug effects , Cell Differentiation/physiology , Cell Proliferation/drug effects , Cells, Cultured , Female , Glial Fibrillary Acidic Protein , Heparin-binding EGF-like Growth Factor , Intercellular Signaling Peptides and Proteins/pharmacology , MAP Kinase Signaling System/drug effects , Male , Mice , Mice, 129 Strain , Mice, Inbred C57BL , Nerve Tissue Proteins/metabolism , Nestin/metabolism , Receptors, Notch/metabolism , Vimentin/metabolism
5.
Tissue Eng Part C Methods ; 20(6): 485-92, 2014 Jun.
Article in English | MEDLINE | ID: mdl-24102451

ABSTRACT

Neuronal signal transduction and communication in vivo is based on highly complex and dynamic networks among neurons expanding in a three-dimensional (3D) manner. Studies of cell-cell communication, synaptogenesis, and neural network plasticity constitute major research areas for understanding the involvement of neurons in neurodegenerative diseases, such as Huntington's, Alzheimer's, and Parkinson's disease, and in regenerative neural plasticity responses in situations, such as neurotrauma or stroke. Various cell culture systems constitute important experimental platforms to study neuronal functions in health and disease. A major downside of the existing cell culture systems is that the alienating planar cell environment leads to aberrant cell-cell contacts and network formation and increased reactivity of cell culture-contaminating glial cells. To mimic a suitable 3D environment for the growth and investigation of neuronal networks in vitro has posed an insurmountable challenge. Here, we report the development of a novel electrospun, polyurethane nanofiber-based 3D cell culture system for the in vitro support of neuronal networks, in which neurons can grow freely in all directions and form network structures more complex than any culture system has so far been able to support. In this 3D system, neurons extend processes from their cell bodies as a function of the nanofiber diameter. The nanofiber scaffold also minimizes the reactive state of contaminating glial cells.


Subject(s)
Hippocampus/cytology , Nanofibers/chemistry , Nerve Net/physiology , Neurons/cytology , Neurons/physiology , Printing, Three-Dimensional , Tissue Scaffolds , Animals , Batch Cell Culture Techniques/instrumentation , Cell Adhesion/physiology , Cell Proliferation/physiology , Cell Survival/physiology , Cells, Cultured , Electroplating/methods , Hippocampus/physiology , Humans , Mice , Mice, Inbred C57BL , Nanofibers/ultrastructure , Nerve Net/cytology , Particle Size , Rotation , Tissue Engineering/instrumentation , Tissue Engineering/methods
6.
Glia ; 61(3): 432-40, 2013 Mar.
Article in English | MEDLINE | ID: mdl-23292921

ABSTRACT

We tested the hypothesis that astrocytes grown in a suitable three-dimensional (3D) cell culture system exhibit morphological and biochemical features of in vivo astrocytes that are otherwise lost upon transfer from the in vivo to a two-dimensional (2D) culture environment. First, we report development of a novel bioactively coated nanofiber-based 3D culture system (Bioactive3D) that supports cultures of primary mouse astrocytes. Second, we show that Bioactive3D culture system maintains the in vivo-like morphological complexity of cultured cells, allows movement of astrocyte filopodia in a way that resembles the in vivo situation, and also minimizes the cellular stress, an inherent feature of standard 2D cell culture systems. Third, we demonstrate that the expression of gap junctions is reduced in astrocytes cultured in a 3D system that supports well-organized cell-cell communication, in contrast to the enforced planar tiling of cells in a standard 2D system. Finally, we show that astrocytes cultured in the Bioactive3D system do not show the undesired baseline activation but are fully responsive to activation-inducing stimuli. Thus, astrocytes cultured in the Bioactive3D appear to more closely resemble astrocytes in vivo and represent a superior in vitro system for assessing (patho)physiological and pharmacological responses of these cells and potentially also in co-cultures of astrocytes and other cell types.


Subject(s)
Astrocytes/cytology , Brain/cytology , Cell Culture Techniques/methods , Animals , Cell Shape , Mice
7.
Blood ; 119(20): 4752-61, 2012 May 17.
Article in English | MEDLINE | ID: mdl-22262761

ABSTRACT

Tissue-type plasminogen activator (t-PA) can modulate permeability of the neurovascular unit and exacerbate injury in ischemic stroke. We examined the effects of t-PA using in vitro models of the blood-brain barrier. t-PA caused a concentration-dependent increase in permeability. This effect was dependent on plasmin formation and potentiated in the presence of plasminogen. An inactive t-PA variant inhibited the t-PA-mediated increase in permeability, whereas blockade of low-density lipoprotein receptors or exposed lysine residues resulted in similar inhibition, implying a role for both a t-PA receptor, most likely a low-density lipoprotein receptor, and a plasminogen receptor. This effect was selective to t-PA and its close derivative tenecteplase. The truncated t-PA variant reteplase had a minor effect on permeability, whereas urokinase and desmoteplase were ineffective. t-PA also induced marked shape changes in both brain endothelial cells and astrocytes. Changes in astrocyte morphology coincided with increased F-actin staining intensity, larger focal adhesion size, and elevated levels of phosphorylated myosin. Inhibition of Rho kinase blocked these changes and reduced t-PA/plasminogen-mediated increase in permeability. Hence plasmin, generated on the cell surface selectively by t-PA, modulates the astrocytic cytoskeleton, leading to an increase in blood-brain barrier permeability. Blockade of the Rho/Rho kinase pathway may have beneficial consequences during thrombolytic therapy.


Subject(s)
Astrocytes/drug effects , Blood-Brain Barrier/drug effects , Blood-Brain Barrier/metabolism , Fibrinolysin/pharmacology , Tissue Plasminogen Activator/pharmacology , rho-Associated Kinases/metabolism , Animals , Astrocytes/metabolism , Astrocytes/physiology , Blood-Brain Barrier/pathology , Blood-Brain Barrier/physiology , Cell Membrane Permeability/drug effects , Cells, Cultured , Coculture Techniques , Dose-Response Relationship, Drug , Drug Evaluation, Preclinical , Enzyme Activation/drug effects , Fibrinolysin/metabolism , Humans , Mice , Mice, Inbred C57BL , Models, Theoretical , Signal Transduction/drug effects , Signal Transduction/physiology
8.
Neurosignals ; 18(3): 152-63, 2010.
Article in English | MEDLINE | ID: mdl-21109727

ABSTRACT

Reactive astrogliosis constitutes a major obstacle to neuronal regeneration and is characterized by rearrangement and upregulation of expression of cytoskeletal proteins, increased proliferation and hypertrophy. Many approaches have been attempted to mimic astrogliosis by inducing reactive astrocytes in vitro. Such research is usually performed using astrocytes derived from Mus musculus or Rattus norvegicus, and results compared between species on the assumption that these cells behave equivalently. Therefore, we compared reactivity between mouse and rat astrocytes in scratch wound assays to gain further insight into how comparable these cell culture models are. Proliferation and migration, as well as expression of the cytoskeletal proteins glial fibrillary acidic protein (GFAP) and vimentin, were compared by immunocytochemistry and immunoblot. Further, we investigated migration of proliferating cells by 5-ethynyl-2'-deoxyuridine staining. Substantial differences in GFAP expression and proliferation between astrocytes of the two species were found: rat astrocytes showed different cytoskeletal morphology, expressed significantly more GFAP and vimentin of different molecular size and were more proliferative than comparable mouse astrocytes. Our results suggest that rat and mouse astrocytes may respond differently to various reactivity-triggering stimuli, which needs to be considered when general conclusions are drawn regarding effects of factors regulating astrocyte reactivity.


Subject(s)
Astrocytes/cytology , Astrocytes/metabolism , Cell Proliferation , Animals , Animals, Newborn , Cells, Cultured , Gliosis/pathology , Gliosis/physiopathology , Mice , Mice, Inbred C57BL , Rats , Rats, Sprague-Dawley , Species Specificity
9.
J Neurochem ; 113(4): 881-94, 2010 May.
Article in English | MEDLINE | ID: mdl-20202079

ABSTRACT

EphA4 null mice have impaired astrocytic gliosis following spinal cord injury. This may be because of altered cytoskeletal regulation and is examined herein using cultured astrocytes from wildtype and EphA4 null mice. Under basal conditions EphA4 null astrocytes appeared relatively normal but following stimuli resulting in cytoskeletal rearrangement, EphA4 null cells responded more slowly. When F-actin stress fibers were collapsed using the Rho kinase inhibitor HA1077, fewer EphA4 null cells showed stress fiber collapse in response to HA1077 and recovered stress fibers more slowly following HA1077 removal. EphA4 null astrocytes were less adherent and had smaller focal adhesions, while activation of Eph receptors with ephrin-A5-Fc increased the numbers of focal adhesions in both wildtype and knockout astrocytes following serum starvation. Using scratch wound assays, EphA4 null astrocytes invading the scratch showed impaired glial fibrillary acidic protein expression, particularly in proliferative cells. Astrocytes did not express Ephexin, a major Eph-interacting Rho guanine exchange factor, but they expressed Vav proteins, with lower levels of phospho-Vav in EphA4 null compared to wildtype astrocytes. This may contribute to the slower cytoskeletal responses generally observed in the EphA4 null astrocytes. Eph receptor signaling therefore regulates astrocyte reactivity through modulation of cytoskeletal responses.


Subject(s)
Astrocytes/metabolism , Cytoskeleton/metabolism , Gliosis/metabolism , Receptor, EphA4/metabolism , Actins/metabolism , Animals , Animals, Newborn , Astrocytes/ultrastructure , Cell Adhesion/physiology , Cell Movement/physiology , Cell Proliferation , Cells, Cultured , Cytoskeleton/ultrastructure , Enzyme Inhibitors/pharmacology , Ephrin-A5/pharmacology , Focal Adhesions/metabolism , Glial Fibrillary Acidic Protein/metabolism , Gliosis/genetics , Gliosis/physiopathology , Mice , Mice, Inbred C57BL , Mice, Knockout , Proto-Oncogene Proteins c-vav/metabolism , Receptor, EphA4/agonists , Receptor, EphA4/genetics , Spinal Cord Injuries/metabolism , Spinal Cord Injuries/physiopathology , rho-Associated Kinases/antagonists & inhibitors , rho-Associated Kinases/metabolism
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