Brain-derived neurotrophic factor (BDNF) is the most prominent neurotrophic factor in the brain that promotes differentiation, maturation, and survival of neurons (Lewin and Barde 1996). In addition, BDNF and its cognate receptor tyrosine kinase TrkB play important roles in synaptic plasticity and higher brain functions, such as learning and memory (Nawa and Takei 2001; Poo 2001). Many of the events that underlie plasticity depend on translation (Costa-Mattioli et al. 2009; Richter and Klann 2009). We have previously shown that BDNF up-regulates translation by activating mammalian target of rapamycin (mTOR) signaling in neurons (Takei et al. 2001, 2004; Inamura et al. 2005). mTOR is a serine/threonine protein kinase that integrates signaling mediated by growth factors/receptor tyrosine kinases, and various nutrients, such as amino acids and glucose. mTOR nucleates two distinct complexes, mTOR complex 1 (mTORC1) and mTOR complex 2. mTORC1 plays a central role in mRNA translation and autophagy, suggesting that mTORC1 regulates total protein levels. Interestingly, recent reports have revealed that mTOR is a key molecule for translation-dependent learning and memory (Belelovsky et al. 2009; Hoeffer and Klann 2010; Qi et al. 2010).
Various nutrients, such as amino acids and glucose, serve as not only raw materials and energy sources for protein synthesis, but also signaling molecules that regulate the translation machinery through mTORC1 (Sengupta et al. 2010). For instance, branched chain amino acids, in particular leucine, activate mTORC1-mediated signaling (Tokunaga et al. 2004; Hara et al. 1998; Ishizuka et al. 2008). Cellular glucose levels, which are closely related to the AMP/ATP ratio, affect mTORC signaling pathway via AMP-activated protein kinase (AMPK) (Inoki et al. 2003; Tokunaga et al. 2004; Kimura et al. 2003). Ligands of receptor tyrosine kinases are known to control translation levels. It has been well studied that insulin and insulin receptor signaling activates the translation machinery through multiple factors, including mTORC1 (Proud and Denton 1997; Proud 2006). These multiple cascades, amino acids, glucose, and receptor tyrosine kinase signals are merged on mTORC1 and interplay, leading to the downstream cellular responses such as regulation of translation (Hay and Sonenberg 2004; Wullschleger et al. 2006; Sengupta et al. 2010; Proud 2007).
Our previous studies found that BDNF plays a key role in neurons to control translation via mTORC1 (Inamura et al. 2005; Takei et al. 2001, 2004). Moreover, leucine activates p70S6 kinase (p70S6K) in neurons in a rapamycin-dependent manner (Ishizuka et al. 2008). The aim of this study was to elucidate the interplay between BDNF and certain nutrients in the central nervous system. Since amino acid(s) sufficiency is reported to be essential for the insulin-induce activation of mTORC1 in PC12 and chinese hamster ovary cells (Kleijn and Proud 2000; Campbell et al. 1999), we examine the inter-related effects between amino acids or glucose and BDNF in primary cultured cortical neurons. Unlike these cell lines, the effects of BDNF on mTORC1 signaling in neurons are dependent on glucose, but not amino acids.
We found that AMPK suppressed the effects of BDNF on mTORC1-dependent translation in neurons. To date, how neurons integrate information about cellular energy status and BDNF–TrkB signaling has remained unclear. Therefore, we analyzed the role of AMPK in BDNF-induced p70S6K a substrate of mTORC1, activation and consequent protein synthesis in neurons.
AMPK is a heterotrimeric serine/threonine protein kinase, consisting of a catalytic α subunit and regulatory β and γ subunits (Hardie 2004). AMPK serves as a cellular energy sensor by detecting the AMP/ATP ratio. When the ratio is elevated – for instance, during periods of metabolic stress from nutrient insufficiency or hypoxia/ischemia – AMPK activates energy-generating catabolic pathways and suppresses such anabolic pathways as fatty acid synthesis, gluconeogenesis, and protein synthesis (Inoki et al. 2003). Of note, AMPK activity is coupled with phosphorylation of Thr172 in the AMPKα subunit (Kemp et al. 2003; Witters et al. 2006; Hardie 2004). Here, we report that AMPK activation abolishes the effects of BDNF on protein synthesis by specifically suppressing activation of mTOR signaling in cortical neurons.
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- Materials and methods
- Supporting Information
This study examined the mutual relationships between BDNF/TrkB signaling and nutrients signals in neurons. Brain function clearly depends on the levels of essential nutrients. A lack of certain nutrients reduces brain activity and leads to a loss of consciousness. Recent studies have shown that amino acids and glucose are not only used as materials for protein synthesis and energy metabolism but also function as signaling molecules. Indeed, in neurons, amino acids – notably, leucine (Ishizuka et al. 2008; Cota et al. 2006) – and glucose (Dash et al. 2006) activate mTORC1 in vitro and in vivo.
In this study, we found differences in the nutrients required by fibroblasts and neurons for growth factor-induced p70S6K phosphorylation at Thr389, a substrate of mTORC1. In PC12 (Kleijn and Proud 2000) and chinese hamster ovary (Campbell et al. 1999) cells, amino acids, but not glucose, are essential for insulin-induced mTORC1 signaling. Although similar results were observed in primary fibroblasts, neurons showed a different profile. BDNF and insulin-induced p70S6K phosphorylation at Thr389 even in the absence of amino acids, whereas the effects of these molecules were largely suppressed under glucose-free conditions. These data raise the question of how glucose deprivation interrupts BDNF/TrkB activation of mTORC1 in neurons.
Several lines of evidence showed that the glucose deprivation protocol used in this study decreased but did not completely deplete ATP levels. Phosphorylation was not generally defected (Fig. 2). Thus, we focused on AMPK because it serves as an intracellular energy sensor that detects decreases in ATP concentrations and increases in AMP levels (Kemp et al. 2003; Witters et al. 2006; Hardie 2004). AMPK activates tuberous sclerosis complex (TSC), GTPase-activating proteins for Rheb, resulting in inhibition of mTORC1 activity (Inoki et al. 2003). After confirming that glucose deprivation and 2DG treatment induced AMPKα phosphorylation at Thr172, that represents enzymatic activation, the effects of other AMPK activators were examined in neurons. The efficacy of these compounds differ among cell type, possibly because their permeability or uptake (Kimura et al. 2003). AICAR, a structural analog of AMP, and the biguanide antidiabetic drug metformin induced AMPKα phosphorylation and activation in cortical neurons, a process that was not related to reduce ATP levels (Corton et al. 1995; Bolster et al. 2002). These agents inhibited BDNF-induced activation of p70S6K in cortical neurons (see also Figs 4 and 7). Further confirmation of the effects of AMPK activity on BDNF-induced mTORC1 signaling was achieved by transfecting neurons with CA-AMPKα. AMPK is a tri-heteromeric enzyme that is composed of a catalytic α subunit and regulatory β and γ subunits. The construct composed of AMPKα1 amino acid residues 1-312 lacked the autoinhibitory domain, resulting in a constitutively active enzyme. In addition, the mutant form is more stable than wild-type AMPKα1 (Crute et al. 1998). Considering the low efficacy of transfection (about 20%), partial inhibition of p70S6K phosphorylation induced by BDNF is quite reasonable. Our results indicate that AMPK activation interferes with BDNF-induced mTORC1 signaling and protein synthesis in neurons. Interestingly, neither BDNF nor insulin suppresses AMPKα phosphorylation (Figure S4). Whereas in vivo experiments showed that insulin suppressed AMPK activity in hypothalamus (Minokoshi et al. 2004), no direct effect was observed in cultured cortical neurons (Figure S4).
It is unclear why neurons depend on glucose but not amino acids in the experiments described here. One possibility is that intracellular levels of free amino acids in neurons may be higher than in other cell types. Alternatively, rates of autophagy in neurons may be fast enough to create a sufficient supply of amino acids for the time window (2 h) of the experiments in this study.
AMPK phosphorylates Thr1271 and Ser1387 of TSC2, thereby activating its GTPase-activating protein activity to Rheb, whereas Akt, a downstream effector of BDNF/TrkB, phosphorylates several other residues and inhibits its activity (Huang and Manning 2008). Although further experiments are required, the results presented here suggest that Thr1271 (and possibly Ser1387) phosphorylation may overcome the effect of Akt-induced phosphorylation to induce the GTPase-activating protein activity of TSC2. Interestingly, AMPK activation by 2DG also suppressed the phosphorylation of Akt at Ser473 induced by BDNF (Figure S2). Because Ser473 is a substrate residue of phosphorylation by mTORC2 (Sarvassov et al. 2005), AMPK may affect the activity of mTORC2 as well, possibly through TSC2.
In addition, AMPK phosphorylates Raptor at Ser722/792 and induces binding to 14-3-3, thus inhibiting mTORC1 activity (Gwinn et al. 2008). In cortical neurons, AMPK activation also induced raptor phosphorylation at Ser 792. The results suggest that AMPK activation attenuated BDNF-induced activation of mTORC1 signaling both through TSC2 and raptor.
Glucose-deprivation or adding 2DG creates a relatively extreme environment, which may mimic pathological conditions, such as ischemia and hypoglycemia induced by hyperinsulinemia. AMPK, however, can be activated under physiologic conditions. Several endogenous molecules regulate AMPK activity in the brain (Lim et al. 2010). For example, ghrelin (Andersson et al. 2004) and adiponectin (Kadowaki et al. 2008) activate AMPK, whereas leptin (Minokoshi et al. 2002) and glucagon-like peptide 1 (Seo et al. 2008) inhibit the enzyme in the brain. These hormones centrally regulate appetite. Interestingly, BDNF is reported to be a strong anorexigenic factor (Pelleymounter et al. 1995), and there may be crosstalk between feeding-related molecules and BDNF in the brain via AMPK and mTORC1.
In addition to eating behaviors, AMPK may contribute to synaptic plasticity (Potter et al. 2010) and other higher brain functions, such as learning and memory, by interfering with the BDNF–mTORC1 signaling pathway. As noted previously, BDNF is critical for learning and memory (Lewin and Barde 1996; Nawa and Takei 2001; Poo 2001). Moreover, mTORC1 plays a central role in translation-dependent learning and memory (Hoeffer and Klann 2010; Qi et al. 2010). Several kinases mediate AMPKα phosphorylation, leading to enzymatic activation (Lim et al. 2010). Among them, calcium/calmodulin-dependent protein kinase kinase β (Hawley et al. 2005; Woods et al. 2005) is primarily expressed in neurons, suggesting an important role in AMPK activation in the brain under physiologic conditions. Regulated by Ca2+, calcium/calmodulin-dependent protein kinase kinase β may transduce signaling from neurotransmitters and neuropeptides to activate AMPK and inhibit the activity of BDNF.