ation experiment demonstrated that mTOR and gephyrin were enriched in the synaptosomal fraction, but not the postsynaptic density fraction (117).
Another possible connection between mTOR signaling and localized translation is via the modulation of eEF2 phosphorylation. Several studies have noted an increase in eEF2 phosphorylation in response to various neurotransmitters. For example, glutamate or NMDA treatment of cortical neurons in culture leads to a rapid and pronounced increase in eEF2 phosphorylation, and a decrease in translation rates in cell bodies and proximal (but not distal) cell processes (121). Activation of the NMDA receptor also leads to eEF2 phosphorylation, in tadpole tecta (122). It is tempting to speculate that mTOR could inhibit eEF2 phosphorylation in active synapses to locally derepress translation. It has also been suggested that eEF2 phosphorylation could actually enhance the translation of specific mRNAs localized to dendrites by driving these mRNAs from untranslated ribonucleotide particles or small polysomes into larger polysomes (122–125).
Another possible link between TOR and neuronal function is the regulation of autophagy. In addition to nutrient scavenging during starvation, autophagy has been demonstrated to play an important role in developmental processes that involve cellular remodeling, such as insect metamorphosis (126) or luteal regression (127). Whereas neuronal death certainly involves apoptosis (128), several reports have suggested that an alternative form of cell death may occur in some nerve cells. For example, nerve growth factor (NGF)-deprivation of sympathetic neurons was reported to induce a rapid, 30-fold increase in autophagic particles, before any signs of DNA fragmentation (a hallmark of apoptosis) were observed. Treatment of these cells with the anti-autophagic drug 3-methyladenine delayed cell death (129). In another study, autophagic vacuoles were observed in PC12 cells 3 h after serum starvation, whereas chromatin condensation did not occur until 6 h poststarvation (130). Finally, the removal of specific spinal cord neurons in Xenopus tadpoles (a normal developmental process during metamorphosis) was also suggested to occur through autophagy-directed cell death (131). Intriguingly, elevated levels of autophagy have been reported to be associated with neurodegenerative disorders such as Parkinson’s disease (132).
A recently described mouse mutant suggests that mTOR plays a critical role in embryonic brain development (133; K.Hentges and A.Peterson, personal communication). The murine flat top mutation was isolated in a chemical mutagenesis screen designed to identify genes involved in embryonic telencephalic development (133). Flat top defects include a failure of the embryo to up-regulate proliferation in the telencephalic primordium, and a failure to establish dorsal and ventral domains of gene expression in the developing telencephalon. Homozygous mutant embryos fail to rotate around the body axis, and die in utero (78). The flat top mutation was mapped to a single nucleotide change in an mTOR intron, which leads to aberrant splicing. The protein products derived from these abnormally spliced mRNAs appear to be inactive (or much less active), because of the presence of a 3-aa insertion or 3-aa deletion at the intron-exon junction. Transgenic rescue experiments confirmed that mTOR is the affected gene in this animal, and a rapamycin injection regimen during pregnancy yields embryos with an identical phenotype (K. Hentges and A.Peterson, personal communication). Whether the brain defect is the result of a failure to inhibit autophagy, or is elicited through some other function of mTOR is unknown. S6K1 activity was demonstrated to be significantly lower (17% of wild-type levels) in flat-top embryos, but effects on other translation factors have not yet been determined. The flat top mouse should provide a very valuable tool for the study of TOR function in mammalian cells.
The TORs are evolutionarily conserved protein kinases that regulate the balance between protein synthesis and degradation in unicellular and multicellular organisms. This complex balance is maintained via the regulation of translation initiation and elongation factor activity, the modulation of ribosome biosynthesis at both the transcriptional and translational levels, the control of amino acid permease activity, the coordination of the transcription of many enzymes involved in various metabolic pathways, and the control of autophagy. An interesting and unexpected finding was that mTOR also appears to play a critical role in embryonic brain development, learning, and memory formation.
There is still much to be learned. For instance, how the TOR proteins sense the quality or quantity of nutrients is unknown. The mammalian GCN2 kinase, which senses intracellular amino acid levels by binding to deacylated tRNAs, does not appear to play a role in this process, because amino acid withdrawal leads to S6K1 and 4E-BP1 dephosphorylation even in GCN2 null cells (C.Jousse and D.Ron, personal communication). Further, whereas the role of the TOR proteins in the control of metabolic enzymes and amino acid permeases in S. cerevisiae is now well documented, similar studies have not been conducted for the mammalian and Drosophila systems. The recent description of the Drosophila TOR homolog (dTOR; refs. 21 and 22) and the isolation of the murine flat top mTOR mutant (133) should provide invaluable tools for further dissection of the TOR signaling module in multicellular organisms.
We thank A.Peterson, K.Hentges, C.Jousse, D.Ron, F.Peiretti, and J.W.B.Hershey for sharing unpublished data, and W.S.Sossin, F. Poulin, P.F.Cho-Park, and M.Miron for critical reading of the manuscript. Work in the authors’ laboratory is supported by grants from the Canadian Institutes of Health Research, the National Cancer Institute of Canada, the Howard Hughes Medical Institute (HHMI), and the Human Frontier Science Program. B.R. is supported by a Medical Research Council (MRC) of Canada postdoctoral fellowship. A.-C.G. is supported by an MRC of Canada doctoral fellowship. N.S. is an MRC of Canada Distinguished Scientist and an HHMI International Scholar.
1. Goelet, P., Castellucci, V.F., Schacher, S. & Kandel, E.R. (1986) Nature (London) 322, 419–422.
2. Bailey, C.H., Bartsch, D. & Kandel, E.R. (1996) Proc. Natl. Acad. Sci. USA 93, 13445–13452.
3. Sossin, W. S. (1996) Trends Neurosci. 19, 215–218.
4. Frey, U. & Morris, R.G. (1998) Trends Neurosci. 21, 181–188.
5. Steward, O. & Levy, W.B. (1982) J. Neurosci. 2, 284–291.
6. Tiedge, H. & Brosius, J. (1996) J. Neurosci. 16, 7171–7181.
7. Torre, E.R. & Steward, O. (1992) J. Neurosci. 12, 762–772.
8. Weiler, I.J. & Greenough, W.T. (1993) Proc. Natl. Acad. Sci. USA 90, 7168–7171.
9. Kang, H. & Schuman, E.M. (1996) Science 273, 1402–1406.
10. Martin, K. C., Casadio, A., Zhu, H., E, Y., Rose, J.C, Chen, M., Bailey, C.H. & Kandel, E.R. (1997) Cell 91, 927–938.
11. Casadio, A., Martin, K.C., Giustetto, M., Zhu, H., Chen, M., Bartsch, D., Bailey, C.H. & Kandel, E.R. (1999) Cell 99, 221–237.
12. Vezina, C., Kudelski, A. & Sehgal, S.N. (1975) J. Antibiot. (Tokyo) 28, 721–726.
13. Harding, M.W., Galat, A., Uehling, D.E. & Schreiber, S.L. (1989) Nature (London) 341, 758–760.
14. Siekierka, J.J., Hung, S.H., Poe, M., Lin, C.S. & Sigal, N.H. (1989) Nature (London) 341, 755–757.
15. Siekierka, J.J., Wiederrecht, G., Greulich, H., Boulton, D., Hung, S.H., Cryan, J., Hodges, P.J. & Sigal, N.H. (1990) J. Biol. Chem. 265, 21011–21015.
16. Cafferkey, R., Young, P.R., McLaughlin, M.M., Bergsma, D.J., Koltin, Y., Sathe, G.M., Faucette, L., Eng, W.K., Johnson, R.K. & Livi, G.P. (1993) Mol. Cell. Biol. 13, 6012–6023.
17. Heitman, J., Movva, N.R. & Hall, M.N. (1991) Science 253, 905–909.