mTOR (mammalian target of rapamycin) signaling pathways

In order to increase the complexity of neuronal connection, the processes of neurites arborization and spine formation were constantly occurred during growth/development. This requires various signal

pathways to interact. For example, members of the Rho family of proteins, Rho A or Cdc 42, direct actin cytoskeleton development, while

Ras-Raf-MAPK pathway involves in accepting extracellular signals into nucleus to modulate dendritic filopodia formation (Wu et al., 2001; Luo, 2002; Alonso et al., 2004). Recently, it was found that mTOR pathway is

also involved in many aspects to regulate the morphological and functional stability of neurons (Garelick and Kennedy, 2011).

mTOR complexes and signaling

mTOR, a serine-threonine protein kinase with multiple domains, belongs to the phosphatidylinositol 3-kinase-related kinases (PIKKs) family (Bosotti et al., 2000). It is responsible for merging extracellular information to govern many cellular processes such as growth, survival, and metabolism (Wullschleger et al., 2006). In mammalian cells, mTOR forms different complexes by associating with different proteins, named mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). The components of mTORC1 are mTOR, mLST8 and raptor, while mTORC2 is comprised of mTOR, mLST8, mSin1 and rictor (Sarbassov et al., 2006).

Two complexes display distinct functions. One of the characterized properties is that mTORC1 is rapamycin-sensitive whereas mTORC2 is rapamycin-insensitive (Jacinto et al., 2004). Rapamycin, produced from Streptomyces hygroscopicus, can bind with FKBP12 to target on mTOR kinase and to selectively inhibit mTORC1 activity (Hara et al., 2002; Kim et al., 2002; Hay and Sonenberg, 2004; Sarbassov et al., 2004). Thus, rapamycin has been used as a strategic approach to elucidate the involvement of mTORC1.

The current model/hypothesis involved in mTORC1 pathway is depicted as follows (Wang and Proud, 2011). By exogenous growth factors or nutrient binds on their specific receptors, the receptors trigger a series of signal cascades such as insulin receptor, insulin receptor

substrate 1, and PI3K by mediating though protein/protein interaction (Hay and Sonenberg, 2004).Phosphatidylinositol-3,4,5-triphosphate (PIP3) generated by activated PI3K will recruit both PDK1 and Akt onto

membrane where PDK1 will phosphorylate Akt on Thr 308. mTORC2 was demonstrated to be able to phosphorylate substrate Akt on Ser 473 site (Sarbassov et al., 2005). Normally, tuberous sclerosis protein 1 (TSC1) and tuberous sclerosis protein 2 (TSC2) form a heterodimeric complex to suppress mTORC1 activity. TSC2 acts as a

GTPase-activating protein (GAP) to enhance GTPase activity which will convert the Rheb into GDP-bound form, an inactive state (Vander Haar et al., 2007). Upon growth factor/nutrient activates the upstream molecules, the TSC1/2 complex will dissociate due to the phosphorylation of TSC2 by activated Akt. This will result in the release of inhibition by which no more GAP activity toward Rheb. This will lead Rheb-GTP to activate mTORC1 (Long et al., 2005).

The downstream of mTORC1 action is well characterized on its ability to regulate translation initiation. It employed phosphorylating S6 kinase (S6K) and eukaryotic initiation factor 4E-binding proteins

(4E-BPs) to control translation and cell cycle (Fingar and Blenis, 2004;

Ekim et al., 2011; Thoreen et al., 2012).

Two isoforms of S6K, S6K1 and S6K2, are both present in

mammalian cells. S6K1 has two isoforms, cytosolic p70 and nuclear p85, while p54 and p56, the two isoforms of S6K2, are in nucleus (Lagasse and Clerc, 1988; Jacinto and Lorberg, 2008).Both of them are belonging to AGC (PKA, PKG and PKC) kinase family, shared with the similar

structure of a small N-terminal lobe and a larger C-terminal lobe to

co-ordinate the binding of ATP. Kinase domains of them are 84% identity but quite differ in N-and C-terminus (Jacinto and Lorberg, 2008). S6K1 has been well known as the target of mTORC1, whereas related

researches on S6K2 are limited. N-terminus of S6K1 executes as receiving active information to suppress the autoinhibitory C-terminus and its TOR signaling (TOS) motif can carry S6K to be closed with Raptor, one component of mTORC1 so that the active sites of Thr 389/Thr 229 acquire the opportunities to be phosphorylated by

mTOR/PDK1 (Pullen and Thomas, 1997; Magnuson et al., 2012). Recent studies utilized S6K1^-/- mice to study the importance of mTORC1 in regulating embryogenesis, which resulted in smaller size than that of wild type, but not cell numbers. Nevertheless, the size of S6K2^-/-mice is almost similar to that of wild type (Shima et al., 1998; Pende et al., 2000; Pende et al., 2004; Russell et al., 2011; Magnuson et al., 2012). Phosphorylation of S6K1 on Thr389 directly promote activate ribosomal protein S6 to phosphorylate eIF4E to promote translation initiation as well. In addition, mORC1 regulates elongation phase via phosphorylated eEF2 (Wang et al., 2001).

The other mTORC1 target, 4E-BP, also has three isoforms and contains one TOS motif in the N terminus to modulate the binding of 4E-BP to raptor for multi-sites phosphorylation. Insulin or serum elevates the phosphorylation of 4E-BP1 on Thr37/46 and thus promotes the

activation on Thr70/Ser65 (Schalm et al., 2003; Eguchi et al., 2006).

Phosphorylated 4E-BP1 would release itself from eIF4E by which eIF4E

is capable to associate with eIF4G and to attract ribosomes loading onto mRNA, initiating the so called cap-dependent translation (Ruvinsky and Meyuhas, 2006) Similarly, 4E-BP1 also functions in controlling cell size without affecting the cell number (Murakami et al., 2004).

mTOR pathway and neurons

mTOR signaling pathway revealed in most cellular types seems to be also observed in neurons. 4E-BP and S6K involved in

mTOR-dependent protein translation were found to present in dendrites close to synaptic sites (Richter and Klann, 2009). Rapamycin, was able to prevent the establishment of L-LTP which requires synapse

protein-dependent enhancement (Hoeffer and Klann, 2010). In addition, mTORC1 signaling also implies key information for hypothalamus to control food intake. S6K1 knockout mice are resistant to high fat diet induced obesity (Um et al., 2004; Cota et al., 2006). Furthermore, Akt had been proved that its deficiency caused significant mental disorder, due to the terminated dopaminergic signaling (Beaulieu et al., 2009). A recent report further confirmed this mTORC2-related mechanism on brain by using rictor knockout mice (Siuta et al., 2010). However, more detail molecular mechanisms of mTOR complexes involved in those situations still remain unclear.

4. EMBRYONIC STEM CELLS AND NEURONAL