ABSTRACT
Specific cell types such as neurons, astrocytes, and microglial cells can be isolated from a mixed population of human foetal brain cells and cultured for extended periods. The rapid expansion of cell populations occurring during culture has accommodated the large-scale growth and production of various neurotropic viruses. Furthermore, neural cell lines derived from CNS tumours or by the immortalization of primary cells have also been established and used for studies of viral pathogenesis. The potential to generate and expand selected populations of neural-derived cells should provide a new and abundant substrate for the production of viruses in vaccine development.
Subject(s)
Astrocytes/cytology , Microglia/cytology , Neurons/cytology , Viral Vaccines , Cell Line, Transformed , Fetus/cytology , HumansABSTRACT
The predominant cell types infected by HIV-1 in AIDS associated encephalopathy are cells of the macrophage/microglial lineage. There has been consistent evidence, however, that astrocytes also become infected although not at the same frequency or level of multiplication as microglial cells. HIV-1 antigens and/or nucleic acid have been identified in astrocytes in brain autopsy tissue from both adult and pediatric AIDS cases. In cell cultures, HIV-1 infection of astrocytes results in an initial productive but non-cytopathogenic infection that diminishes to a viral persistence or latent state. Understanding the nature of HIV-1 infection of astrocytes, which represents the largest population of cells in the brain, will contribute to the understanding of AIDS encephalopathy and the dementia that occurs in nearly one-quarter of all AIDS patients.
Subject(s)
Astrocytes/virology , Brain/virology , HIV Infections/virology , HIV-1 , Astrocytes/metabolism , Brain/pathology , Cells, Cultured , Fetus , Fluorescent Antibody Technique , HIV Infections/pathology , HumansABSTRACT
The presence of the intermediate filament protein nestin has been the predominant marker used to describe stem and progenitor cells in the mammalian CNS. In this study, a 998-bp fragment in the 3' region of the nestin mRNA was cloned from human fetal brain cells (HFBC). The nucleotide sequence of the cloned cDNA revealed 21 differences with the previously published human nestin sequence, resulting in 17 amino acid changes. A 150-amino-acid fragment derived from the cloned nestin cDNA was coupled to glutathione S-transferase and used as an immunogen to generate a rabbit polyclonal antiserum that selectively detects human nestin. HFBC that proliferated in response to basic fibroblast growth factor incorporated 5-bromo-2'-deoxyuridine into their nuclei and immunostained for nestin, indicating nestin expression in proliferating CNS progenitor cells. In all cell cultures, nestin costained with the neuroepithelial cell marker vimentin. A small subset of nestin-stained cells (1-2%) immunostained with neuronal marker MAP-2 during the first week and after 4 weeks in culture. However, during the first week in culture, approximately 10-30% of the total cell population of HFBC stained for the glial cell marker GFAP, and nearly all coimmunostained for nestin. After 4 weeks in culture, a subset of GFAP-positive cells emerged that no longer costained with nestin. These results describe nestin expression not only in CNS progenitor cells but also in the cells which were in transition from a progenitor stage to glial differentiation. Collectively, these data suggest a differential temporal regulation of nestin expression during glial and neuronal cell differentiation.
Subject(s)
Brain/metabolism , Intermediate Filament Proteins/genetics , Nerve Tissue Proteins , Neuroglia/metabolism , Neurons/metabolism , Amino Acid Sequence , Animals , Antibody Specificity , Brain/cytology , Brain/embryology , Cell Line , Cloning, Molecular , Humans , Immunoblotting , Immunohistochemistry , Intermediate Filament Proteins/analysis , Intermediate Filament Proteins/chemistry , Molecular Sequence Data , Nestin , Neuroglia/cytology , Neurons/cytology , RNA, Messenger/genetics , Rabbits , Rats , Recombinant Proteins/chemistry , Reverse Transcriptase Polymerase Chain Reaction , Sequence AlignmentABSTRACT
Time lapse video microscopy is used to study the chronology of morphological changes and commitment to die in individual PC12 cells after induction of apoptosis. Cell death is highly asynchronous occurring over a 2- to 3-day period following serum removal; however, all cells go through three characteristic morphological phases irrespective of the time they die following serum removal. During phase 1, which lasts from 2 to 44 h, cells maintain normal morphology. Phase 2 is characterized by plasma membrane bubbling which lasts from 10 min to 40 h. Phase 3 represents the active or execution phase of apoptosis and involves dynamic whole cell body blebbing. Phase 3/execution phase has a restricted duration, lasting 96 +/- 5 min. At the end of the execution phase of apoptosis, cells die. The inherently asynchronous nature of cell death is still present in cells that are synchronized following mitosis. Daughter cells enter phase 2 synchronously but remain in phase 2 for varying periods and die at different times. Addition of serum 24-48 h after initiating apoptosis blocks death of 89% of cells in phase 1, 79% in phase 2, and 0% in phase 3. Serum rescue experiments are consistent with cells committing to die about 2-3 h prior to the onset of phase 3 (execution phase of apoptosis). These studies indicate that although apoptosis is an asynchronous process it can be defined in terms of reproducible morphological changes that can be used to place other events, such as the commitment to die, in a temporal sequence.
Subject(s)
Apoptosis/physiology , Animals , Cell Survival , Culture Media, Serum-Free , Microscopy, Video , PC12 Cells , Rats , Time FactorsABSTRACT
The effects of intrastriatal injections of a reversible inhibitor of succinate dehydrogenase, malonate, on the extracellular concentrations of amino acid neurotransmitters were examined using a microdialysis probe that was positioned a fixed distance from an injection cannula. Malonate (2 mumol) caused a 23 +/- 5-fold increase in extracellular glutamate (Glu), a 18 +/- 6-fold increase extracellular gamma-aminobutyric acid (GABA) and a modest increase in extracellular aspartate (Asp, 2.9 +/- 0.8-fold increase). Administration of the NMDA receptor antagonist MK-801 (5 mg/kg) prior to injection of malonate almost completely blocked these increases. This study provides direct evidence that inhibition of succinate dehydrogenase causes an increase in extracellular amino acid neurotransmitters and further evidence that bioenergetic defects may contribute to the pathogenesis of chronic neurodegenerative diseases through an excitotoxic mechanism.