Faculty Bibliography

Neurotrophins, such as NGF and BDNF, activate Trk receptor tyrosine kinases through receptor dimerization at the cell surface followed by autophosphorylation and intracellular signaling. It has been shown that activation of Trk receptor tyrosine kinases can also occur via a G-protein-coupled receptor (GPCR) mechanism, without involvement of neurotrophins. Two GPCR ligands, adenosine and pituitary adenylate cyclase-activating polypeptide (PACAP), can activate Trk receptor activity to increase the survival of neural cells through stimulation of Akt activity. To investigate the mechanism of Trk receptor transactivation, we have examined the localization of Trk receptors in PC12 cells and primary neurons after treatment with adenosine agonists and PACAP. In contrast to neurotrophin treatment, Trk receptors were sensitive to transcriptional and translational inhibitors, and they were found predominantly in intracellular locations particularly associated with Golgi membranes. Biotinylation and immunostaining experiments confirm that most of the transactivated Trk receptors are found in intracellular membranes. These results indicate that there are alternative modes of activating Trk receptor tyrosine kinases in the absence of neurotrophin binding at the cell surface and that receptor signaling may occur and persist inside of neuronal cells

Schwann cell factor 1 (SC1), a p75 neurotrophin receptor-interacting protein, is a member of the positive regulatory/suppressor of variegation, enhancer of zeste, trithorax (PR/SET) domain-containing zinc finger protein family, and it has been shown to be regulated by serum and neurotrophins. SC1 shows a differential cytoplasmic and nuclear distribution, and its presence in the nucleus correlates strongly with the absence of bromodeoxyuridine (BrdU) in these nuclei. Here, we investigated potential transcriptional activities of SC1 and analyzed the function of its various domains. We show that SC1 acts as a transcriptional repressor when it is tethered to Gal4 DNA-binding domain. The repressive activity requires a trichostatin A-sensitive histone deacetylase (HDAC) activity, and SC1 is found in a complex with HDACs 1, 2, and 3. Transcriptional repression exerted by SC1 requires the presence of its zinc finger domains and the PR domain. Additionally, these two domains are involved in the efficient block of BrdU incorporation by SC1. The zinc finger domains are also necessary to direct SC1's nuclear localization. Lastly, SC1 represses the promoter of a promitotic gene, cyclin E, suggesting a mechanism for how growth arrest is regulated by SC1

OBJECT: 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