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Apoptosis, often also termed "programmed cell death", occurs in normal development in the brain and spinal cord. Important to concepts of disease and potential intervention is the exciting finding that apoptosis is also found after neurotrauma and in a number of neurodegenerative diseases. Although the precise mechanism of neuronal cell loss remains unknown, much emphasis has been placed recently on the activation of cell death protease cascades within the cell. How these cascades may be activated, especially from extracellular influences, is currently poorly understood. Thrombin, the multifunctional coagulation protease, is an early phase modulator at sites of tissue injury and has been shown to induce cell death in neurons by an apoptotic mechanism by activating its receptor, PAR-1. Using a model motor neuronal cell line, NSC19, which we have shown undergoes apoptosis after treatment with classic apoptosis inducers such as the topoisomerase inhibitors camptothecin and etoposide, we unambiguously found that nanomolar thrombin induced characteristic signs of apoptosis. Strikingly, endonucleolysis was accompanied by an increase in caspase-3-like activity in cellular extracts, which correlated with both detection of caspase-induced signature cleavage of the cortical cytoskeleton component nonerythroid spectrin (alpha-fodrin) and identification of increased accessibility of a caspase cleavage domain, using an antibody (Ab127) made against a synthetic peptide KGDEVD. Demonstrating that thrombin activation of death proteases was linked to cell death, we were able to inhibit thrombin-induced apoptosis by using a caspase family inhibitor, benzyloxycarbonyl-Asp-(oMe)-fluoromethyl ketone (Boc-D-FMK). These novel results demonstrate that thrombin serves as an extracellular "death signal" to activate intracellular protease pathways. These pathways lead to apoptotic cell death and can be modulated by inhibiting caspase activity downstream to PAR-1.

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.

Study Design. Serum withdrawal was introduced to a spinal cord motor neuron cell line to investigate the mode of cell death. Objectives. To characterize the death of motor neurons in culture, to gain insight into mechanisms that could be important in spinal cord diseases. Summary of Background Data. Normal reduction of cell number during central nervous system development is brought about by programmed cell death. These same apoptotic processes probably play a role in a variety of central nervous system disorders, including traumatic injury. Although certain proteolytic processes are involved, the molecular details involved in the apoptotic induction have not been fully elucidated. Methods. To identify apoptosis, several criteria were used, including analysis of chromatin condensation with DNA-specific stains (propidium iodide and Hoechst 33342); in situ end-labeling of DNA fragments in apoptotic nuclei with terminal deoxynucleotidyl transferase; fragmentation of DNA separated on agarose gel electrophoresis; and cleavage of a characteristic substrate for apoptotic proteases, α-fodrin, into signature cleavage fragments. Results. The NSC19 cell line exhibited motor neuron characteristics morphologically, with typical cellular structure, and biochemically, by synthesizing choline acetyl transferase. Under various treatments including serum withdrawal (loss of trophic factors), cell loss occurred through an apoptotic cell death pathway. Conclusions. A murine motor neuron cell line, NSC19, has been used to investigate apoptosis in this in vitro system. Cell death occurs by apoptosis, suggesting that this cell line may provide a useful model for studying apoptotic mechanisms in spinal cord degeneration and injury.

Loss of intracellular calcium homeostasis has been regarded an important factor underlying neuron cell death after cerebral ischemic insult. In the brain, a major mechanism for regulation of intracellular calcium is through the signal transduction pathway involving hydrolysis of poly-phosphoinositides and release of the second messenger, inositol 1,4,5-trisphosphate (IP3). IP3 mobilizes calcium by interacting with an intracellular receptor. Upon its release after agonist stimulation, this second messenger is catabolized by a 3-kinase and a 5-phosphatase. In this study, in situ hybridization was carried out to examine the mRNA expression of IP3, receptor (IP3R) and IP3 3-kinase (IP3K) in rat brain cortex after transient focal cerebral ischemia induced by temporary occlusion of the middle cerebral artery (MCA) and the common carotid arteries (CCAs). Results indicate a large decrease (52%) in IP3R mRNA levels in the ischemic cortex as compared to that in the contralateral side at 4 h after a 45 min ischemic insult. By 16 h, practically no IP3R mRNA could be detected in the ischemic cortex. On the other hand, IP3K mRNA levels remained unaltered until 16 h after reperfusion, during which time, expression in the infarct core decreased but that surrounding the core area increased instead. Hybridization of adjacent brain sections with probes for neuron specific enolase (NSE) and beta-actin indicated also a time-dependent decrease in mRNA levels after ischemia, but these changes were less dramatic as compared to IP3R. At 16 and 24 h after reperfusion, there was an increase in beta-actin mRNA in cortical areas outside the MCA cortex, suggesting of reactive gliosis.(ABSTRACT TRUNCATED AT 250 WORDS)