Involvement of TLCK-sensitive serine protease in colchicine-induced cell death of sympathetic neurons in culture.

Abstract

Superior cervical ganglion (SCG) cells from neonatal rats underwent apoptosis upon treatment with colchicine, a microtubule-disrupting agent. Western blotting and activity measurements showed that caspase-3 was indeed activated, but its peptide inhibitor (Ac-DEVD-CHO) neither suppressed nuclear fragmentation nor rescued the neurons from cell death. z-VAD-fmk, the general inhibitor of caspases, prevented nuclear fragmentation and delayed the cell death. Moreover, N-alpha-tosyl-L-lysine chloromethyl ketone (TLCK), but not N-alpha-tosyl-L-phenylalanine chloromethyl ketone (TPCK), prevented nuclear fragmentation and provided neuronal protection as well. The combination of both z-VAD-fmk and TLCK provided a long-term neuronal protection (>4 days), whereas neither one alone could do so, suggesting that there are both caspase-dependent and -independent pathways. TLCK-sensitive serine protease is also likely to act upstream of caspase-3 in a caspase-dependent pathway. Electron microscopic observations demonstrated that z-VAD-fmk suppressed nuclear fragmentation and improved mitochondrial swelling, but failed to prevent vesicular formation, which resulted in a slowly-occurring necrosis. More importantly, TLCK effectively blocked this abundant vesicular formation along with suppressing chromatin condensation. Thus, the combination of both conferred a nearly normal morphology, which is consistent with the results of cell survival experiments. These findings clearly indicate that TLCK-sensitive serine protease plays multiple roles in caspase-dependent and -independent pathways of colchicine-induced cell death, and suggest a novel mechanism underlying a necrotic pathway involving ER swelling and vesicular formation.

• Authors: C Mitsui, K Sakai, T Ninomiya, T Koike
• Journal: Journal of neuroscience research (J Neurosci Res) Vol. 66 Issue 4 Pg. 601-11 (Nov 15 2001) ISSN: 0360-4012 [Print] United States
• PMID: 11746380 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: Copyright 2001 Wiley-Liss, Inc.

Chemical References

• Enzyme Inhibitors
• Microtubule Proteins
• Neuroprotective Agents
• Protein Synthesis Inhibitors
• Serine Proteinase Inhibitors
• Tosyllysine Chloromethyl Ketone
• Tosylphenylalanyl Chloromethyl Ketone
• Bucladesine
• Serine Endopeptidases
• Casp3 protein, rat
• Caspase 3
• Caspases
• Potassium
• Colchicine

Topics

• Animals
• Animals, Newborn
• Apoptosis (drug effects, physiology)
• Bucladesine (pharmacology)
• Caspase 3
• Caspases (drug effects, metabolism)
• Cells, Cultured (drug effects, enzymology, ultrastructure)
• Colchicine (pharmacology)
• Enzyme Inhibitors
• Immunohistochemistry
• Microscopy, Electron
• Microtubule Proteins (antagonists & inhibitors, metabolism)
• Necrosis
• Nervous System (drug effects, enzymology, ultrastructure)
• Neurons (drug effects, enzymology, ultrastructure)
• Neuroprotective Agents (pharmacology)
• Potassium (pharmacology)
• Protein Synthesis Inhibitors (pharmacology)
• Rats
• Rats, Sprague-Dawley
• Serine Endopeptidases (drug effects, metabolism)
• Serine Proteinase Inhibitors (pharmacology)
• Signal Transduction (drug effects, physiology)
• Superior Cervical Ganglion (drug effects, enzymology, ultrastructure)
• Tosyllysine Chloromethyl Ketone (pharmacology)
• Tosylphenylalanyl Chloromethyl Ketone (pharmacology)