Malcolm Schinstine

Expression of neuronal antigens by astrocytes derived from EGF-generated neuroprogenitor cells.

Previous studies have demonstrated that astrocytes reacting to CNS injury can express antigens normally associated with neurons. The origin of the reactive astrocytes, i.e., whether they are newly differentiated glial cells or preexisting astrocytes somehow triggered to express neuronal markers, remains difficult to determine using an in vivo model system. An in vitro model may prove more manageable. In the present study, primary brain cultures and EGF-generated neuroprogenitor cells were used to study the expression of neuronal antigens by established (primary) and nascent astrocytes, respectively. Astrocytes derived directly from dissociated mouse brains exhibited a flat morphology typical of type 1 astrocytes. These cells were nestin and GFAP positive and, in most cases, the antigens were colocalized. Primary astrocytes did not appear to express the putative neuronal markers GABA, Tau, or MAP2. Nascent astrocytes derived from EGF-generated progenitor cells showed a similar pattern of GFAP and nestin immunoreactivity. Contrary to primary astrocytes, many GFAP-intensive, stellate astrocytes exhibited Tau and MAP2. These cells also exhibited an intense nestin immunoreactivity. These data suggest that the reactive astrocytes expressing neuronal antigens in response to CNS trauma may be derived from neural progenitor cells rather than from previously differentiated astrocytes.

5-Azacytidine and BDNF enhance the maturation of neurons derived from EGF-generated neural stem cells

EGF-generated neural stem cells can form astrocytes, neurons, and oligodendrocytes upon differentiation; however, the proportion of cells that actually form neurons is very small. In the present study, we have studied the effect that 5-azacytidine (5AzaC), a demethylation agent, and brain-derived growth factor (BDNF) have on the differentiation and maturation of neurons originating from EGF-generated neural stem cells. Stem cells were maintained under a variety of culture conditions using combinations of 5AzaC and BDNF either alone or together. More neurons, as determined by the number of beta-tubulin III-immunoreactive somata, were present in cultures maintained in BDNF medium (a nearly fourfold increase compared to control cultures). 5AzaC did not significantly affect neuronal number, regardless of the presence of BDNF. In addition to neuronal number, the effect of 5AzaC and BDNF on the distribution of the microtubule proteins MAP2 and Tau was analyzed. In most cultures, MAP2 and Tau were colocalized throughout the neuron. In contrast, neurons cotreated with 5AzaC and BDNF contained neurons that began to exhibit cytoskeletal segregation of MAP2 into the somatodendritic compartments. Tau remained dispersed within the somata and the axon. This effect was not produced when 5AzaC or BDNF was used individually. These results demonstrate that 5AzaC and BDNF cooperate to produce more mature neurons from EGF-generated neural stem cells then either molecule can alone.

Effects of Choline and Quiescence on Drosophila Choline Acetyltransferase Expression and Acetylcholine Production by Transduced Rat Fibroblasts

Abstract: Rat‐1 fibroblasts were transduced to express Drosophila choline acetyltransferase. The presence of an active enzyme in these cells (Rat‐1/dChAT) was confirmed using various methods. Rat‐1/dChAT fibroblasts released acetylcholine (ACh) into the culture medium. Moreover, intra‐and extracellular levels of ACh could be increased by adding exogenous choline chloride. In addition, serum starvation or confluence‐induced quiescence caused an 80% decrease in recombinant choline acetyltransferase activity (compared with actively growing cells). ACh release was also repressed in quiescent fibroblast cultures. Exogenous choline could mitigate the decrease in ACh release. These results indicate that Rat‐1 fibroblasts can be genetically modified to produce ACh and that ACh release can be controlled by introducing choline into the culture medium. Furthermore, these data demonstrate that the expression of the retroviral promoter used in this study decreases with the onset of quiescence; however, exogenous choline can increase the amount of ACh released by quiescent fibroblasts.

Potential effect of cytokines on transgene expression in primary fibroblasts implanted into the rat brain

Fibroblasts genetically modified with retroviral vectors fail to demonstrate long-term transgene expression upon implantation into the body. Although the mechanisms behind this phenomenon have not been elucidated, one likely cause is the response of the host to the graft. For example, genetically modified fibroblasts grafted into the brain are surrounded by activated microglia and astrocytes. The apparent inflammatory response can last for several weeks. In addition, the center of the graft is typically infiltrated with macrophage-like cells that appear to reside continuously within the graft. This proximity of inflammatory cells to the graft suggests that these cells may somehow influence transgene expression. In the current study, an in vitro model was used to test the effect cytokines [transforming growth factor-beta1 (TGF-beta1), interleukin-1beta, (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha)] that are typically released by peripheral macrophages, activated microglia and/or astrocytes have on long-terminal repeat (LTR)-driven transgene expression in primary fibroblasts. Our data demonstrate that these cytokines can significantly reduce the steady-state level of proviral mRNA. The amount of proviral mRNA returned to control levels within 24 h if the cytokines were removed. In addition, the down-regulation of proviral mRNA levels could be prevented if the cells were incubated with dexamethasone (25 microM) concurrent with the introduction of cytokines. These data demonstrate that cytokines can down-regulate LTR-driven transgene expression in primary fibroblasts maintained in culture. This interaction may be a major reason why transduced cells do not demonstrate long-term transgene expression in vivo.

Intracerebral delivery of growth factors: Potential application of genetically modified fibroblasts

To date, a number of different growth factors (e.g. nerve growth factor, brain-derived neurotrophic factor, neurotrophin-3, ciliary neurotrophic factor, and fibroblast growth factor) have been shown to act as a neurotrophic and/or neurotrophic agents on distinct neuronal populations within the peripheral and central nervous system. Knowledge as to how most of these factors influence the development and regeneration of growth factor-sensitive neurons has been obtained from in vitro examination. A new approach that can be used to assess the effects of growth factors on neuronal groups in vivo is the combined use of gene transfer and intracerebral grafting techniques. The present article explores the potential use of grafting genetically modified fibroblasts within the nervous system as a delivery method for growth factors.

Chapter 26. Action of Neurotrophic Factors on Central Nervous System Neurons

Publisher Summary The study of neurotrophic factors is still relatively young. A neurotrophic factor is a molecule produced by various cell types that can affect the neuronal growth. Agents that affect neuronal survival, the size of the cell body (hypertrophy), neurite (axon or dendrite) elongation, and neurotransmitter synthesis can be considered as neurotrophic factors. Evidence demonstrating a role for various molecules during regeneration and development suggest a possible clinical application for these types of molecules. For example, it has been suggested that nerve growth factor (NGF) be used as a potential therapy for neurodegenerative diseases that involve cholinergic neurons, such as been described in Alzheimers. Although some neurotrophic factors influence many aspects of neuronal growth, others have a limited effect—that is, maintaining survival or promoting neurite outgrowth only. The functions of four neurotrophic factors are discussed in this chapter with major emphasis on the nerve growth factor (NGF). The theme of the chapter is restricted to the effects of the described agents on central nervous system (CNS) neurons during development and after injury in the adult. The best characterized neurotrophic factor is undoubtedly NGF. The vast majority of NGF-sensitive neurons in the CNS are cholinergic; however, some evidence now suggests that embryonic GABAergic neurons are also NGF-responsive. Several other neurotrophic factors that may potentially prove important are gangliosides, hormones, such as thyroid hormone, interleukins, and potentially, various neurotransmitters. These molecules have been shown to influence the neuronal survival, cytoarchitecture, and the regeneration of various CNS neuronal populations. Till date, however, it is still early to employ these drugs therapeutically. Much more basic research, especially on how trophic factors elicit a response from target neurons, is needed.