Apoptotic, injury-induced cell death in cultured mouse murine motor neurons

doi:10.1016/S0304-3940(97)00468-0

Neuroscience Letters

Volume 230, Issue 1, 11 July 1997, Pages 25-28

https://doi.org/10.1016/S0304-3940(97)00468-0 Get rights and content

Abstract

In order to develop in vitro models of CNS injury, astrocytes have been mechanically injured in culture to study reactive astrocytosis. However, scratch injury models of pure neuronal cultures have not yet been exploited to study programmed cell death (PCD). For this study, we examined model motor neurons (NSC19 cells) in culture and found time-dependent cell death in proximity (within 2.5 mm) to a physical scratch injury. Injury-induced cell death was apoptotic verified by positively-stained nuclei using both the in situ end-labeling (ISEL) procedure and Hoechst 33342. Unexpectedly, cells proximal to the injury site were not affected by the injury until 3 days later suggesting that adjacent motor neuron loss was dependent on a `death signal' produced by direct injury to sister neurons. `Executioners' in apoptosis include free radicals, cell cycle kinases and cysteine proteases (caspases). Extracellular serine proteases, such as thrombin and granzyme B, may activate such intracellular pathways and several inhibitors (serpins), such as CrmA, are effective in blocking apoptosis. Since protease nexin I (PNI), a serpin homologous with CrmA, prevents apoptosis of lumbar motor neurons and is increased after nerve injury, we examined mRNA by RT-PCR for PNI expression. Of interest, although we were unable to find significant levels of PNI message in NSC19 cells, we did detect it in the parent neuroblastoma.

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Acknowledgements

We thank Ray Salcedo and Keith Ratzlaff for expert technical assistance, Dr. Neil Cashman for providing the NSC19 cells, and Dr. Dennis Wallace for statistics discussion. This study was supported, in part, by the Jackson Foundation, USUHS (DVHIP), the ALS and Spinal Cord Injury Fund of The Kansas University Endowment Association, The Research Institute, Inc., of the University of Kansas Medical Center, and the Medical Research Service of the Department of Veterans Affairs.

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Antibody to murine PAR1 was a generous gift of Drs. Patricia Andrade-Gordon and Michael D’Andrea (R. W. Johnson Pharmaceutical Research Institute, Spring House, PA). As in our previously-published studies (Citron et al., 1997; Smirnova et al., 1998a; Smirnova et al., 1998b), we used motor neuron-like NSC19 (or NSC34) cells (initially a gift from Dr. N. Cashman, Montreal Neurologic Hospital, Montreal, Canada), a mouse-mouse neural hybrid cell line produced through fusion of the aminopterin-sensitive neuroblastoma N18TG2 with motor neuron-enriched embryonic day 12-14 spinal cord cells, (Cashman et al., 1992). Relevant to our experiments, a prior report utilized these same cells to estimate the effects of HNE on glutamate transport (Pedersen et al., 1999).
Thrombin and membrane lipid peroxidation (MLP) have been implicated in various central nervous system (CNS) disorders from CNS trauma to stroke, Alzheimer's (AD) and Parkinson's (PD) diseases. Because thrombin also induces MLP in platelets and its involvement in neurodegenerative diseases we hypothesized that its deleterious effects might, in part, involve formation of MLP in neuronal cells. We previously showed that thrombin induced caspase-3 mediated apoptosis in motor neurons, via a proteinase-activated receptor (PAR1). We have now investigated thrombin's influence on the oxidative state of neurons leading to induction of MLP-protein adducts. Translational relevance of thrombin-induced MLP is supported by increased levels of 4-hydroxynonenal-protein adducts (HNEPA) in AD and PD brains. We now report for the first time that thrombin dose-dependently induces formation of HNEPA in NSC34 mouse motor neuron cells using anti-HNE and anti-acrolein monoclonal antibodies. The most prominent immunoreactive band, in SDS-PAGE, was at ∼54 kDa. Membrane fractions displayed higher amounts of the protein-adduct than cytosolic fractions. Thrombin induced MLP was mediated, at least in part, through PAR1 since a PAR1 active peptide, PAR1AP, also elevated HNEPA levels. Of interest, glutamate and Fe₂SO₄ also increased the ∼54 kDa HNEPA band in these cells but to a lesser extent. Taken together our results implicate the involvement of thrombin and MLP in neuronal cell loss observed in various CNS degenerative and traumatic pathologies.
VEGF protects rat cortical neurons from mechanical trauma injury induced apoptosis via the MEK/ERK pathway
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In several studies, the survival effects of VEGF in primary neuronal cultures are mediated by the anti-apoptotic pathway involving the MEK/ERK pathway [21,27]. Mechanical trauma injury induced cell death with apoptotic features in cultured neurons [4]. Our study indicates that pretreatment with VEGF prevented scratch-induced neuronal death.
Traumatic brain injury (TBI) is a serious insult that frequently leads to neurological dysfunction or death. Vascular endothelial growth factor (VEGF) is a major regulator of angiogenesis and vascular permeability. Recently, VEGF has been identified as a neurotrophic factor and has been implicated in the pathogenic mechanisms of TBI. However, the possible mechanisms of VEGF in primary or secondary injuries after TBI are largely unknown. The present study attempted to determine whether VEGF has a protective effect on primary cortical neurons against mechanical trauma injury, which is an in vitro insult mimicking traumatic brain injury. We found that pretreatment of primary cortical neurons in culture with VEGF decreased neuronal death in a concentration-dependent manner, and VEGF counteracted the mechanical trauma mediated apoptotic death of cultured cortical neurons. VEGF up-regulates the activity of ERK (extracellular signal-regulated kinase) in cultured cortical neurons and U0126 (a mitogen activated protein kinase kinase (MEK) inhibitor) suppressed VEGF induced activity of ERK. Furthermore, incubation of cells with U0126 attenuated the ability of VEGF to protect neurons against mechanical trauma-induced apoptosis. Therefore, the present study supports the notion that MEK/ERK pathway is involved in VEGF mediated neuroprotection against mechanical trauma injury.
Differentially expressed genes of the carpet shell clam Ruditapes decussatus against Perkinsus olseni
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Further investigations are needed to elucidate this aim, and will be the objective of a separate publication. Apoptosis or programmed cell death (that essentially acts on the elimination of unwanted cells [5,14]) can play an important role in the progression of disease, and normally occurs when a cell is damaged, infected or senescent [45] and also plays an important role during the elimination of harmful cells [14]. The DAD-1 (defender against apoptotic cell death) is involved, among other processes, in the genetic control of apoptosis inhibiting it [33], and it is known to be a highly conserved gene among humans, mice, clawed frogs, plants, and even recently have been characterized in arachnids, and in the marine invertebrate A. irradians [53] and S. purpuratus (GenBank accession number XP001178079) suggesting that this gene could have similar biochemical functions in animal and plants.
Suppression–Subtractive Hybridization (SSH) was used to identify differentially expressed Ruditapes decussatus genes against the protozoan Perkinsus olseni infection. A forward and a reverse subtraction were carried out to identify up- and down-regulated genes in both haemocytes and gills of clams naturally infected with P. olseni. New genes, candidates for further investigation into the functional basis of resistance to pathogens, have been detected for the first time in the clam (R. decussatus). A total of 305 differentially expressed sequences were obtained, 221 of them in haemocytes and 84 in gills of infected clams. The number of ESTs with potential similarity with known genes was 97, 42 among them were related with immunity and stress related functions. The pattern of expression of the immune selected genes was studied by quantitative PCR with samples of naturally Perkinsus infected clams and compared with samples from an in vitro infection of clam haemocytes with Perkinsus zoospores. The maximum expression was found 1 h post infection. The complete open reading frames of selected sequences (Rd adiponectin-C1q and Rd DAD-1) were determined. Our results provide new insights into the molecular basis of host–pathogen interactions in R. decussatus.
Tissue transglutaminase during mouse central nervous system development: Lack of alternative RNA processing and implications for its role(s) in murine models of neurotrauma and neurodegeneration
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Tissue transglutaminase (tTG) is a member of a multigene family principally involved in catalyzing the formation of protein cross-links. Unlike other members of the transglutaminase family, tTG is multifunctional since it also serves as a guanosine triphosphate (GTP) binding protein (Gα_h) and participates in cell adhesion. Different isoforms of tTG can be produced by proteolysis or alternative splicing. We find that tTG mRNA is expressed at low levels in the mouse CNS relative to other tissues, and at lower levels in the CNS of mouse in comparison to that of human or rat. tTG mRNA levels are higher in the heart compared to the CNS, for example, and much higher in the liver. Within the CNS, tTG message is lowest in the adult cerebellum and thalamus and highest in the frontal cortex and striatum. In the hippocampus, tTG expression is highest during embryonic development and falls off dramatically after 1 week of life. We did not find alternative splicing of the mouse tTG. At the protein level, the predominant isoform is ∼62 kDa. In summary, tTG, an important factor in neuronal survival, is expressed at low levels in the mouse CNS and, unlike rat and human tTG, does not appear to be regulated by alternative splicing. These findings have implications for analyses of rodent tTG expression in human neurodegenerative and neurotrauma models where alternative processing may be an attractive pathogenetic mechanism. They further impact on drug discovery paradigms, where modulation of activity may have therapeutic value.
Keratinocyte Growth Factor 1 Inhibits Wound Edge Epithelial Cell Apoptosis In Vitro
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Previously, it was found using mouse motor neurons in culture that sister cells undergo apoptosis adjacent to those at the “epicenter” of the scratch injury. This was restricted only to those neurons proximal to the scratch, early after injury, suggesting that a “death signal” or wave of apoptosis is produced by injured cells moving outward from the injury and not by factors diffusing from the wound edge cells (Citron et al, 1997). Signaling processes have also been detected radiating from scrape-wounded intestinal epithelial cell cultures.
The ability of keratinocyte growth factor 1 to modulate apoptosis in the absence of proliferation was studied in vitro. A HaCaT scrape wound model was developed in which dense monolayers prior to wounding were cultured to quiescence in defined media with hydroxyurea at concentrations that blocked proliferation without loss of cell viability. Scrape wounding was then found to induce apoptosis, originating at the wound edge, but subsequently radiating away over a 24 h period to encompass areas not originally damaged. Keratinocyte growth factor 1 inhibited this radial progression of apoptosis in a concentration-dependent manner up to 20 ng per mL with induced migration present at the wound edge. The extent of this rescue was modulated by the concentration of Ca²⁺ prior to wounding. In control wound cultures apoptotic bodies were found in cells adjacent to the wound interface but were greatly reduced in keratinocyte-growth-factor-1-treated groups. Keratinocyte growth factor 1 receptor expression was significantly induced within two to three cell widths of the scraped wound edge, at levels far exceeding those found at the leading edge of a nonwounded epithelial sheet. Tumor necrosis factor α (1–5 ng per mL) or Escherichia coli lipopolysaccharide (10–50 ng per mL) exacerbated scrape-induced early apoptosis (1–4 h), but was largely ameliorated by coculture with keratinocyte growth factor 1. Keratinocyte growth factor 1 protection was associated with a reduction in both caspase-3 activation and cytokeratin-19 loss. Protected wound edges were also associated with the maintenance of e-cadherin expression and induction of β1 integrin and actin stress fiber organization. These results suggest that keratinocyte growth factor 1 may play a role in limiting mechanically induced apoptotic processes at the epithelial wound edge in a manner that is distinct from its proliferative function.
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Apoptotic, injury-induced cell death in cultured mouse murine motor neurons

Abstract

Section snippets

Acknowledgements

Prog. Brain Res.

Neuroscience

Int. J. Dev. Neurosci.

Trends Neurosci.

Cell

Neural apoptosis

Ann. Neurol.

Neuroblastoma x spinal cord (NSC) hybrid cell lines resemble developing motor neurons

Dev. Dynamics

DNA strand breaks induced by sustained glutamate excitotoxicity in primary neuronal cultures

J. Neurosci.

Characterization of apoptosis in cultured rat sympathetic neurons after nerve growth factor withdrawal

J. Cell Biol.

Involvement of wound-associated factors in rat brain astrocyte migratory response to axonal injury: in vitro simulation

J. Clin. Invest.