Prevention of apoptotic but not necrotic cell death following neuronal injury by neurotrophins signaling through the tyrosine kinase receptor.
OBJECTIVE
Neurotrophins prevent the death of neurons during embryonal development and have potential as therapeutic agents. During development, neuronal death occurs only by apoptosis and not by necrosis. Following injury, however, neurons can die by both processes. Data from prior studies have not clearly indicated whether neurotrophins can decrease apoptosis compared with necrosis. The goal of this study was to determine the effect of neurotrophin treatment on each of these processes following injury and to characterize the receptor(s) required.


METHODS
The authors used an in vitro model of injury with the aid of primary cortical neurons obtained from rat embryos. After 9 days in culture and the elimination of glia, homogeneous and mature neurons were available for experimentation. Noxious stimuli were applied, including radiation, hypoxia, and ischemia. Subsequent cell death by apoptosis or necrosis was noted based on morphological and enzymatic assessments (such as lactate dehydrogenase [LDH] release) and assays for DNA fragmentation. The effect of treatment with nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 was determined. Finally, Western blot analyses were performed to note the neurotrophin receptor status in the neurons (tyrosine kinase receptors [Trks] and p75). The authors studied different stimuli-induced cell death by using different processes. With the application of radiation, cells died primarily by apoptosis, as evidenced by cell shrinkage, the presence of apoptotic bodies, and specific DNA fragmentation. This was a delayed process (> 6 hours) that could be reduced by gene transcription or protein synthesis inhibitors. With ischemia, cells died immediately by necrosis, showing cell enlargement and rupture. Ischemic cell death was not affected by the inhibition of macromolecular synthesis. Hypoxia produced a mixture of the two cell death processes. Both BDNF and neurotrophin-3 demonstrated protection against apoptotic cell death only. Statistically significant decreases of both LDH release and apoptosis-specific DNA fragmentation were noted following radiation and hypoxia, but not for ischemia. Nerve growth factor, unlike the other neurotrophins, did not affect apoptosis because a functional receptor, Trk A, was not expressed by the cortical neurons. There was expression of both Trk B and Trk C, which bind BDNF and neurotrophin-3.


CONCLUSIONS
These findings have significant clinical implications. Neurotrophins may only be effective in disorders in which apoptosis, and not necrosis, is the major process. Furthermore, the Trk signaling cascade must be activated for this response to occur. Because the expression of these receptors diminishes in adulthood, neurotrophin application may be most appropriate in the pediatric population.
Kim
Dong H
DH
Department of Neurosurgery, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA. dkim7@partners.org
Zhao
Xiurong
X
Tu
Christina H
CH
Casaccia-Bonnefil
Patrizia
P
Chao
Moses V
MV
D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology* / Q000528:radiation effects; D019208:Brain-Derived Neurotrophic Factor / Q000494:pharmacology; D015687:Cell Hypoxia; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology; D005260:Female; D066298:In Vitro Techniques; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D009336:Necrosis; D020932:Nerve Growth Factor / Q000494:pharmacology; D009414:Nerve Growth Factors / Q000494:pharmacology*; D009474:Neurons / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D020933:Neurotrophin 3 / Q000494:pharmacology; D011247:Pregnancy; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D020813:Receptor, trkB / Q000378:metabolism*; D020812:Receptor, trkC / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects*; D014158:Transcription, Genetic / Q000187:drug effects
D019208:Brain-Derived Neurotrophic Factor; D009414:Nerve Growth Factors; D020933:Neurotrophin 3; D011500:Protein Synthesis Inhibitors; D020932:Nerve Growth Factor; D007770:L-Lactate Dehydrogenase; D020813:Receptor, trkB; D020812:Receptor, trkC