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. 1998 Sep 1;18(17):6882-91.
doi: 10.1523/JNEUROSCI.18-17-06882.1998.

Thrombin perturbs neurite outgrowth and induces apoptotic cell death in enriched chick spinal motoneuron cultures through caspase activation

Affiliations

Thrombin perturbs neurite outgrowth and induces apoptotic cell death in enriched chick spinal motoneuron cultures through caspase activation

V L Turgeon et al. J Neurosci. .

Abstract

Increasing evidence indicates several roles for thrombin-like serine proteases and their cognate inhibitors (serpins) in normal development and/or pathology of the nervous system. In addition to its prominent role in thrombosis and/or hemostasis, thrombin inhibits neurite outgrowth in neuroblastoma and primary neuronal cells in vitro, prevents stellation of glial cells, and induces cell death in glial and neuronal cell cultures. Thrombin is known to act via a cell surface protease-activated receptor (PAR-1), and recent evidence suggests that rodent neurons express PAR-1. Previously, we have shown that the thrombin inhibitor, protease nexin-1, significantly prevents neuronal cell death both in vitro and in vivo. Here we have examined the effects of human alpha-thrombin and the presence and/or activation of PAR-1 on the survival and differentiation of highly enriched cultures of embryonic chick spinal motoneurons. We show that thrombin significantly decreased the mean neurite length, prevented neurite branching, and induced motoneuron death by an apoptosis-like mechanism in a dose-dependent manner. These effects were prevented by cotreatment with hirudin, a specific thrombin inhibitor. Treatment of the cultures with a synthetic thrombin receptor-activating peptide (SFLLRNP) mimicked the deleterious effects of thrombin on motoneurons. Furthermore, cotreatment of the cultures with inhibitors of caspase activities completely prevented the death of motoneurons induced by either thrombin or SFLLRNP. These findings indicate that (1) embryonic avian spinal motoneurons express functional PAR-1 and (2) activation of this receptor induces neuronal cell degeneration and death via stimulation of caspases. Together with previous reports, our results suggest that thrombin, its receptor(s), and endogenous thrombin inhibitors may be important regulators of neuronal cell fate during development, after injury, and in pathology of the nervous system.

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Figures

Fig. 1.
Fig. 1.
Examples of cultured chick spinal motoneurons stained with Islet-1 (A) or SC-1 (B) antibodies 24 hr after plating. Motoneuron counts typically showed 83 and 75% of cells positive for Islet-1 and SC-1, respectively, in enriched cultures. Black arrowsindicate Islet-1– (A) or SC-1– (B) positive motoneurons. The white arrowhead in (A) points to an Islet-1–negative cell, whereas the double white arrowsshow a dying neuron. Scale bar, 25 μm.
Fig. 2.
Fig. 2.
Motoneuron survival (mean ± SEM) in 48 hr cultures after treatment with different concentrations of either human α-thrombin (A), inactive FSLLRN, or active SFLLRNP (B). Agents were added 2 hr after the initial plating, and cultures were examined for survival 48 hr after the initial plating time. Untreated control motoneurons were grown in L-15 media, and positive controls were treated with soluble chick skeletal muscle extracts (CMX) at 14 μg/ml (data not shown). *p < 0.05; **p < 0.01 versus control; ***p < 0.01 versus 100 μm; n = 3 separate experiments performed in triplicate.
Fig. 3.
Fig. 3.
A–D, Photomicrographs of 2-d-old motoneurons cultured in L-15 media alone (control) (A) or in L-15 media supplemented with 100 nm thrombin (B), 100 nmhirudin (C), or 100 nm hirudin and 100 nm thrombin (D). Note that the thrombin-treated motoneuron exhibits unbranched neurites (B), whereas the cells treated with either hirudin alone (C) or hirudin and thrombin (D) appear similar to the control (A). The inset in Bshows degenerating motoneurons (arrowheads) after treatment with thrombin. Scale bar, 60 μm. E, Surviving motoneuron numbers (mean ± SEM) in 48 hr cultures after treatment with either 100 nm thrombin, 100 nmhirudin, or combinations of these agents at different times. Thrombin and/or hirudin were added to cell cultures at eithert = 0 or t = 24 hr, and all the cultures were examined 48 hr after the initial plating. Cultures treated with thrombin alone at t = 0 (**p < 0.01) or t = 24 hr (*p < 0.05) were significantly different from controls. Cotreatment with hirudin completely prevented thrombin-induced death of motoneurons, whereas addition of hirudin att = 24 hr only partially saved the cells treated with thrombin at t = 0. The short horizontal lines represent no addition of either thrombin or hirudin. ***p < 0.001 and #p < 0.01 versus thrombin treatment at t = 0 andt = 24 hr, respectively.
Fig. 4.
Fig. 4.
A, Mean neurite length (± SEM) per motoneuron in 48 hr cultures after treatment with different concentrations of thrombin (1–1000 nm). Thrombin was added 2 hr after the initial plating, and the cultures were stained with anti-β-tubulin antibodies 48 hr after the initial plating, as described in Materials and Methods. Control cultures were kept in L-15 media. B, The number of primary branches (mean ± SEM) that occurred on the longest neurite in the chick motoneuron cultures at 48 hr after treatment with thrombin. ***p ≤ 0.001 for thrombin treatment versus control in A and B; n = 100 motoneurons examined per group.
Fig. 5.
Fig. 5.
A–D, Photomicrographs of cultured chick spinal motoneurons taken at 60× magnification after TUNEL cytochemistry. A, A negative control culture in which the motoneurons (arrows) were grown solely in L-15 media and were TUNEL-labeled after 6 hr. B, Positive control cells grown in complete medium and treated with 1 mg/ml DNase I for 10 min before TUNEL labeling to produce DNA fragmentation.C, TUNEL-positive nuclei of motoneurons 6 hr after incubation with 100 nm thrombin. D, A TUNEL-positive motoneuron nucleus 18 hr after incubation with 100 nm thrombin. E, Percentage of TUNEL-positive motoneurons at 12 hr after culture in L-15 media alone (control) or with 100 nm thrombin. Approximately 30% of the control cells were TUNEL-positive, whereas, after thrombin treatment, the number of labeled cells increased to 50%. Data are mean ± SEM from three different experiments; ***p < 0.001 for thrombin treatment versus control.
Fig. 6.
Fig. 6.
Electron micrographs depicting a healthy control motoneuron from a culture treated with 14 μg/ml CMX (A) and cultured motoneurons at 12 hr after treatment with 100 nm thrombin (B,C). In B, the plasma membrane, cytosol, and organelles have all been incorporated into apoptotic vesicles (a), and all that remain are the nucleus, which has become small and rounded with an eccentrically placed nucleolus (N), and chromatin condensation (single arrows) around the nuclear membrane.C shows a larger apoptotic vesicle in the process of forming smaller apoptotic vesicles. The apparently poor membrane preservation of the cells has been suggested to be inherent to glutaraldehyde-fixed free (cultured) cells, in contrast to tissue samples (i.e., Robinson et al., 1987). The presence of nonmembrane-bound particles (double arrows inB, C) released in the culture media may be attributable to the lack of phagocytic activity. Scale bars:A, C, 185 nm; B, 14 nm.
Fig. 7.
Fig. 7.
Motoneuron numbers (mean ± SEM) in 48 hr control cultures (open bar) and in cultures treated with either 100 nm thrombin (hatched bars), 1 μm YVAD-CHO, or 10 μm DEVD-CHO (cross-hatched bars) or cotreated with thrombin and either caspase inhibitor (black bars). Agents were added 2 hr after plating, and cells were counted 48 hr after the initial plating. Control cultures were grown in L-15 media. Treatment with 100 nm thrombin significantly decreased cell survival (**p < 0.01 vs control), whereas treatment with either YVAD-CHO or DEVD-CHO alone increased motoneuron survival by 180% (***p < 0.001 vs control). However, the decrease in motoneuron survival after 100 nm thrombin treatment was completely prevented by cotreatment with either YVAD-CHO or DEVD-CHO (#p < 0.001 vs 100 nmthrombin treatment); n = 3 experiments performed in triplicate.

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