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C E Milligan

Publications and source records attributed to C E Milligan.

15 recordsLinked to original sources
PubMed1998

Increased production of amyloid precursor protein provides a substrate for caspase-3 in dying motoneurons.

Biochemical and molecular mechanisms of neuronal cell death are currently an area of intense research. It is well documented that the lumbar spinal motoneurons of the chick embryo undergo a period of naturally occurring programmed cell death (PCD) requiring new gene expression and activation of caspases. To identify genes that exhibit changed expression levels in dying motoneurons, we used a PCR-based subtractive hybridization protocol to identify messages uniquely expressed in motoneurons deprived of trophic support as compared with their healthy counterparts. We report that one upregulated message in developing motoneurons undergoing cell death is the mRNA for amyloid precursor protein (APP). Increased levels of APP and beta-amyloid protein are also detected within dying motoneurons. The predicted peptide sequence of APP indicates two potential cleavage sites for caspase-3 (CPP-32), a caspase activated in dying motoneurons. When peptide inhibitors of caspase-3 are administered to motoneurons destined to undergo PCD, decreased levels of APP protein and greatly reduced beta-amyloid production are observed. Furthermore, we show that APP is cleaved by caspase-3. Our results suggest that differential gene expression results in increased levels of APP, providing a potential substrate for one of the cell death-activated caspases that may ultimately cause the demise of the cell. These results, combined with information on the toxic role of APP and its proteolytic by-product beta-amyloid, in the neurodegenerative disease Alzheimer's, suggest that events of developmental PCD may be reactivated in early stages of pathological neurodegeneration.

Amino Acid Sequence↗
PubMed1998

A novel strategy for introducing exogenous bcl-2 into neuronal cells: the Cre/loxP system-mediated activation of bcl-2 for preventing programmed cell death using recombinant adenoviruses.

We have established a novel strategy for introducing exogenous Bcl-2 into neuronal cells that is mediated by Cre/loxP recombination using recombinant adenoviral vectors. An on/off-switching cassette for Bcl-2 (CALNLbcl-2) was designed to express Bcl-2 by recombinase Cre-mediated excisional deletion of a spacer DNA flanked by a pair of loxP sites. Exogenous Bcl-2 was clearly induced in PC12 cell lines carrying CALNLbcl-2 after infection with recombinant adenovirus producing recombinase Cre (AxCANCre). Dual infection with both AxCANCre and a recombinant adenovirus bearing CALNLbcl-2 showed efficient delivery of exogenous Bcl-2 into a hybrid motoneuronal cell line and primary chicken spinal motoneurons. The delivery of foreign Bcl-2 promoted survival of motoneurons in medium either containing or lacking trophic support. Thus, this strategy for delivery of exogenous Bcl-2 will be useful for studying neuronal death as well as for introducing foreign genes into postmitotic neurons under the control of recombinase Cre.

Adenoviridae↗
PubMed1998

Involvement of specific caspases in motoneuron cell death in vivo and in vitro following trophic factor deprivation.

The caspases have been shown to be key components of programmed cell death (PCD) in various cell types, including neurons. Caspase-3 (CPP32) is the predominant caspase that appears to be involved in cell death in several systems. In embryonic motoneuron cultures, caspase-3 activity increases beginning at 20 h following deprivation of trophic support, as determined by the cleavage of its specific substrates. Inhibition of caspase-3 by peptide inhibitors prevents the PCD of motoneurons following trophic factor deprivation in vitro, as well as in vivo. We also investigated the cleavage of poly(ADP-ribose) polymerase (PARP) in motoneurons after trophic factor withdrawal. No PARP cleavage was detected in either viable or dying cells. These data suggest that some components of the cell death machinery such as the involvement of caspases may be conserved in different cell types undergoing PCD, whereas the activation and specific substrates of the caspases may differ from one cell type to another.

Animals↗
PubMed1997

Cloning and expression of the cDNA encoding rat caspase-2.

We have isolated cDNA clones for rat caspase-2 (also called Nedd2/Ich-1), that encodes a protein similar to interleukin-1beta-converting enzyme (ICE) and the product of the nematode Caenorhabditis elegans cell death gene ced-3 both of which play an important role in programmed cell death (PCD). The rat caspase-2 cDNA clones have an open reading frame (ORF) of 452 amino acids (aa). The predicted aa sequence of rat caspase-2 is highly similar to that of mouse Nedd2 (97.3%) and human Ich-1L (91.3%). The aa sequence QACRG containing the active Cys residue, that is necessary for the proteolytic activity of ICE/Ced-3 (caspase) family of proteases, is also conserved in rat caspase-2. Rat caspase-2 also has several Asp residues in the amino and carboxyl cleavage regions similar to other caspase family proteins. We have developed PC12 cells carrying an on/off switching cassette of caspase-2 (named PC-Nd cells), which contains the neo gene flanked by a pair of loxP sites, the Cre-specific recognition sequence of 34 nucleotides (nt), that lies between the promoter and the caspase-2 cDNA. This expression cassette was designed to express the neo gene initially and to turn on the expression of caspase-2 by site-specific recombinase Cre-mediated excisional deletion of the neo gene. After infection with Cre-producing recombinant adenovirus (re-Ad), the expression of caspase-2 was highly induced in PC-Nd cells and presumptive actively processed fragments of caspase-2 were also observed. This gene activation strategy of caspase-2 will be useful for the study of the biological effects of caspase family proteins in PCD.

Amino Acid Sequence↗
PubMed1997

Programmed cell death during animal development.

The formation of the hand during embryogenesis, the peeling of sunburned skin and the tremor associated with Parkinson's disease all result from a common process: cell death. Cell death occurs throughout the life span of the organism and represents the ultimate differentiative decision made by cells. Insight into the process of cell death will not only contribute to our understanding of basic developmental issues, but will also facilitate the development of therapeutic interventions that could alter the course of disease. Since all cells have the genetic machinery required to commit suicide, the ability to initiate it in a lineage-specific, non-inflammatory manner would allow for the irradication of specific cancers. Alternatively, inhibition of cell death pathways could rescue valuable but condemned cells, such as HIV infected CD4+ T cells or dopaminergic neurons in Parkinson's disease. The goal of this chapter is to provide both an overview of the basic principles that govern the cellular and molecular mechanisms mediating cell death, as well as serve as a reference of known examples of PCD and the genes that mediate this process.

Animals↗
PubMed1997

Identification of a phylogenetically conserved Sug1 CAD family member that is differentially expressed in the mouse nervous system.

We have isolated a cDNA clone from mouse, m56, that encodes a member of the Conserved ATPase-containing Domain (CAD) protein family. Sequence analysis revealed that m56 is identical to mouse mSug1/FZA-B and shares high homology with human Trip1, moth 18-56, and yeast Sug1. When examined, Sug1-like CAD proteins appear to function in the regulation of the 26S proteasome, as well as associate with members of the steroid/thyroid receptor superfamily and other transcriptional activators. m56 can complement the lethal phenotype of loss of SUG1 in yeast. We have examined the tissue distribution of m56 using Northern and Western blots, in addition to immunocytochemistry and in situ hybridization. While m56 was expressed in all tissues and cells examined, several classes of neurons, most notably in the hippocampus, olfactory bulb, and cerebellum, displayed elevated levels of m56 mRNA and protein. We also examined distribution of RNA polymerase II and 26S proteasome subunit 4 (S4) within the mouse brain by in situ hybridization. While all three genes had similar patterns of expression, there were significant differences among them. In moths, the expression of the Sug1 homolog 18-56 is dramatically up-regulated during programmed cell death. In addition, it has been previously demonstrated that the proteasome plays an essential role in the regulation of apoptosis in mammals. We examined the expression of m56 in mouse during natural and induced cell death in a variety of tissues and found no significant changes in expression. Taken together, the data presented here suggest that while m56 is a highly conserved gene that presumably plays essential but complex roles in basal and developmental processes, it may not represent a rate-limiting step in these processes.

ATPases Associated with Diverse Cellular Activitie↗
PubMed1996

Cold thoughts of death: the role of ICE proteases in neuronal cell death.

While there has been extensive work describing the timing, location and probable signals responsible for regulating programmed cell death (PCD) in the nervous system, relatively little is known about the molecular mechanisms that mediate this process. Several investigators have demonstrated that PCD in general, and neuronal PCD in particular, can be inhibited by drugs that arrest RNA or protein synthesis. These data have been interpreted as suggesting that de novo gene expression is required for cells to commit suicide. The general picture emerging from a number of experimental systems is that a variety of proteins can mediate the coupling of extracellular signals to a resident cell-death program. In this model, some of the components required for death are more or less constitutively present in the cell and await lineage-specific signals for their activation. A recent flood of papers has presented convincing evidence that the resident program for apoptosis in numerous cell types works via a series of essential proteases belonging to the CED-3/ICE family.

Animals↗
PubMed1995

Induction of opioid receptor-mediated macrophage chemotactic activity after neonatal brain injury.

Macrophages have a prominent role in the injury response of the brain, yet the molecular mechanisms that control their invasion to the site of neuronal degeneration is unknown. After removal of the posterior cortex at birth, there is massive and specific targeting of nonresident macrophages to axotomized neurons in the lateral thalamus. The present study has identified an injury-induced, brain-derived chemotactic factor (BDCF) capable of eliciting chemotactic responses from resident peritoneal macrophages and brain macrophages. Conditioned media collected from tissue slices containing the axotomized central nervous system neurons exhibit BDCF activity. Initial experiments indicated that BDCF is a small peptide and, thus, we used specific pharmacologic reagents to characterize further BDCF activity. Naloxone, a pan opioid receptor antagonist, completely blocks BDCF activity. Although both kappa and mu opioid receptor antagonists failed to modify BDCF-induced macrophage chemotaxis, two specific delta receptor antagonists blocked BDCF. Analysis of BDCF by reverse phase HPLC and RIA revealed peak chemotactic activity in fractions consistent with the presence of an opioid peptide. The results suggest that cells in the brain respond to neuronal injury by producing and releasing opioids that can initiate a specific macrophage response.

Animals↗
PubMed1995

Apolipophorin III is dramatically up-regulated during the programmed death of insect skeletal muscle and neurons.

The intersegmental muscles (ISMs) of the tobacco hawkmoth Manduca sexta, participate in the emergence behavior of the adult moth and then die during the subsequent 30 hours. In addition, several populations of interneurons and uniquely identified motor neurons also die after adult emergence. The trigger for all of these deaths is a decline in the circulating titer of the insect molting hormone 20-hydroxyecdysone. The ability of the muscles and neurons to die requires de novo gene expression. A differential hybridization screen of a "condemned" ISM cDNA library permitted the isolation of clones encoding four new up-regulated mRNAs. On sequencing, one of these recombinants was found to encode apolipophorin III (apoLp-III), a component of lipophorin, the major hemolymph lipoprotein of insects, previously shown to be synthesized in fat body. Although apoLp-III mRNA and protein were expressed at all stages of ISM development, levels of both molecules were dramatically elevated with the commitment of the cells to die. When ISM cell death was delayed by injection of 20-hydroxyecdysone, expression of apoLp-III at both the RNA and protein levels was markedly reduced at the normal time of cell death. Immunocytochemistry demonstrated that apoLp-III protein was abundantly expressed in the cytoplasm of dying muscles, interneurons, and identified motor neurons at the time of cell death. Apolipoproteins I and II, required components of lipophorin, were not expressed at detectable levels in the muscles or neurons. Furthermore, Western blots of native gels suggest that apoLp-III was not associated with any other proteins. These data suggest that apoLp-III has activities independent of lipid transport that may play a role in programmed cell death. ApoLp-III joins apolipoproteins E and J (clusterin, sulfated glycoprotein-2) as a group of proteins that function in both lipid transfer and cell death.

Animals↗
PubMed1995

Peptide inhibitors of the ICE protease family arrest programmed cell death of motoneurons in vivo and in vitro.

Members of the CED-3/interleukin-1 beta-converting enzyme (ICE) protease family have been implicated in cell death in both invertebrates and vertebrates. In this report, we show that peptide inhibitors of ICE arrest the programmed cell death of motoneurons in vitro as a result of trophic factor deprivation and in vivo during the period of naturally occurring cell death. In addition, interdigital cells that die during development are also rescued in animals treated with ICE inhibitors. Taken together, these results provide the first evidence that ICE or an ICE-like protease plays a regulatory role not only in vertebrate motoneuron death but also in the developmentally regulated deaths of other cells in vivo.

Amino Acid Sequence↗
PubMed1994

Motoneurons deprived of trophic support in vitro require new gene expression to undergo programmed cell death.

During normal development, large numbers of neurons die by programmed cell death. This phenomena has been extensively studied in the lateral motor column of chick embryos, where approximately 50% of the motoneurons that are initially produced, subsequently die due in part to competition for a limited supply of target-derived trophic support. Inhibitors of RNA and protein synthesis block this cell loss in vivo, indicating a requirement for new gene expression (Oppenheim et al., 1990). Prior to their commitment to death, motoneurons can be isolated as a relatively pure population from chick spinal cord for in vitro study. Cells plated with muscle extract, a potent source of target-derived trophic support, survive, and have large, phase-bright cell bodies and extensive neurite outgrowth. In contrast, motoneurons cultured in the absence of muscle extract die within 48 h. This death can be blocked by the RNA synthesis inhibitor actinomycin D, at the time when the cells become committed to die, suggesting that new gene expression is required for cell death. DNA fragmentation and nuclear condensation indicate that some of these cells die by apoptosis. Therefore, it appears that many aspects of motoneuron development observed in vivo can be reconstituted in vitro. These cultures can be used as a model system for studying neuronal death and may contribute to an understanding of the molecular mechanisms that mediate programmed cell death during neuronal development.

Animals↗
PubMed1991

Differential immunochemical markers reveal the normal distribution of brain macrophages and microglia in the developing rat brain.

Brain macrophages and microglia play important roles in central nervous system (CNS) development, especially during regressive events in which particular neuronal and glial constituents are eliminated. The purpose of this study is to provide a complete map of brain macrophage and microglia distribution in all regions of the neuraxis from birth to sexual maturity. We have utilized morphology and immunostaining with the specific antibodies OX-42 and ED1 to distinguish between brain macrophages and microglia. Brain macrophages are large, round cells, 10-15 microns in diameter, with few or no cytoplasmic processes; these cells are ED1- and OX-42-immunopositive. Microglia have small cell bodies with numerous, ramified cytoplasmic processes. These cells are OX-42-positive, and ED1-negative. We found a specific pattern of distribution of brain macrophages, targeting specific cortical and subcortical areas transiently, including developing fiber tracts. These cells disappeared completely by the third postnatal week. In contrast, OX-42-positive microglia exhibited a gradual increase in number and were distributed uniformly throughout gray matter and within white matter tracts. These cells remain in the adult CNS, constituting the resident microglia population. We suggest that these two distinct phagocytic cell populations perform unique functions in the developing brain, including remodeling of restricted CNS areas by brain macrophages that is part of a normal morphological process.

Animals↗
PubMed1991

Brain macrophages and microglia respond differently to lesions of the developing and adult visual system.

Traumatic injury in the brain usually results in rapid degeneration of neuronal elements and a response by peripherally derived macrophages (brain macrophages, BMOs) and resident microglia. One intriguing result of lesions performed in the developing brain as compared to lesions of the mature brain is the faster resolution of the cellular debris and the absence of significant scarring. The purpose of this study was to examine the response of BMOs to induced cell death distant to the lesion site and to investigate possible differences in the responding phagocytic populations (BMOs versus microglia) following lesions in neonates and adults. Ablation of the visual cortex at birth results in very rapid retrograde degeneration and removal of neurons of the dorsal lateral geniculate nucleus (dLGN) within a few days. Lesions to the visual cortex of adult rats also induce neurons within the dLGN to die, but these cells do so over a much more protracted time course. Utilizing differences in morphology and immunocytochemical staining with the monoclonal antibodies ED1 and OX-42 to distinguish between BMOs and microglia, we found that in the developing CNS, BMOs are signalled rapidly and specifically to the location of induced cell death. Microglia are not involved in this response. As might be expected, the temporal response in the adult is much more protracted. In contrast to the developing brain, microglia and not macrophages are the predominant responding cell class after the adult lesion. The data suggest that these are distinct populations of phagocytic cells that respond to brain damage during development and in the adult, which may be critical in modulating the resolution and growth response after injury.

Animals↗
PubMed1991

Persistence of fluoro-gold following degeneration of labeled motoneurons is due to phagocytosis by microglia and macrophages.

When the neural tracer Fluoro-Gold is used to retrogradely label a population of axotomized neurons, cellular labeling can persist in the axotomized nucleus even when Nissl staining indicates that the injured neurons have degenerated. In order to determine the identity of the labeled cells that remain, this study combines retrograde transport of Fluoro-Gold with immunocytochemical methods for identification of specific non-neuronal cell types following peripheral axotomy and Fluoro-Gold labeling of motoneurons in the dorsal motor nucleus of the vagus in neonatal and adult rats. Fourteen days following cervical vagotomy in neonatal rats, Nissl staining revealed a virtually complete loss of vagal motoneurons. Fourteen days after cervical vagotomy in adult rats, vagal motoneuronal loss was not yet extensive but chromatolysis had clearly begun. Injection of Fluoro-Gold into the vagus nerve just prior to the vagotomy led to Fluoro-Gold labeling of remaining vagal motoneurons. In addition, many other small, brightly labeled cells were present in the lesioned vagal nuclei of all rats. Immunofluorescent identification of astrocytes with anti-glial fibrillary acidic protein and microglia and macrophages with OX42 (anti-C3bi complement receptor) and ED1 (anti-monocyte/macrophage cytoplasmic antigen) demonstrated that the small, bright Fluoro-Gold-labeled cells were non-neuronal, non-astrocytic phagocytes, including microglia. These results indicate that phagocytic microglia and other macrophages sequester Fluoro-Gold in the axotomized dorsal motor nucleus of the vagus of neonatal and adult rats, leading to persistence of fluorescent cellular labeling following the loss of retrogradely labeled axotomized neurons.

Age Factors↗