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Nerve growth factor (NGF) is a trophic and survival factor for cholinergic neurons, and it induces the expression of several genes that are essential for synthesis and storage of acetylcholine (ACh), specifically choline acetyltransferase, vesicular ACh transporter (VAChT), and choline transporter. We have found previously that the phosphatidylinositol 3′-kinase pathway, but not the MEK/MAPK pathway, is the mediator of NGF-induced cholinergic differentiation. Here we demonstrate, in the rat pheochromocytoma cell line PC12 and in primary mouse neuronal cultures, that NGF-evoked up-regulation of these three cholinergic-specific genes is mediated by the anti-apoptotic signaling molecule Akt/protein kinase B. Inhibition of Akt activation by the pharmacological inhibitor 1L-6-hydroxymethyl-chiro-inositol 2(R)-2-O-methyl-3-O-octadecylcarbonate (HIMO), or by a peptide fragment derived from the proto-oncogene TLC1, eliminated NGF-stimulated increases in cholinergic gene expression, as demonstrated by RT-PCR and reporter gene assays. Moreover, treatment with HIMO reversed NGF-evoked increases in choline acetyltransferase activity and ACh production. In co-transfection assays with the reporter construct, a dominant-negative Akt plasmid and Akt1-specific small interfering RNA also attenuated NGF-induced cholinergic promoter activity. Our data indicate that, in addition to its well-described role in promoting neuronal survival, Akt can also mediate signals necessary for neurochemical differentiation.
Nerve growth factor (NGF)-TrkA signaling is important for the proper development and functioning of the cholinergic system (Yuen et al. 1996). In addition to its role as a trophic and survival factor for cholinergic neurons, NGF regulates the expression of several genes that are essential for synthesis and storage of acetylcholine (ACh), specifically choline transporter (CHT), choline acetyltransferase (ChAT), and the vesicular ACh transporter (VAChT) (Li et al. 1995; Tian et al. 1996; Pongrac and Rylett 1998; Berse et al. 1999, 2005; Oosawa et al. 1999; Szutowicz et al. 2004). We have been studying the intracellular signaling pathways that mediate these biological actions of NGF. NGF stimulates the MEK/MAPK pathway and this signaling is important for cholinergic function in vivo, as the suppression of extracellular signal-regulated kinase-mediated NGF signaling correlates with cholinergic deficits in aging (Williams et al. 2006, 2007). However, we demonstrated that induction of cholinergic gene expression occurs when the MEK/MAPK pathway is blocked (Madziar et al. 2005). In contrast, pharmacological inhibition of phosphatidylinositol 3′-kinase (PI3K) prevents NGF-induced cholinergic promoter activity, ChAT, VAChT, and CHT mRNA accumulation, and ACh production (Berse et al. 2005; Madziar et al. 2005). Thus, the PI3K pathway is required for the expression of essential cholinergic markers. However, the downstream effectors of PI3K involved in this process remain undetermined.
P13K can phosphorylate both lipids and proteins, but the majority of research focuses on its biological activity as a lipid kinase (Cantley 2002). The phosphorylated lipid products of PI3K function as docking sites to recruit to the membrane many proteins containing pleckstrin homology (PH) domains, including Akt/protein kinase B (Akt/PKB) and phosphoinositide-dependent kinase 1 (PDK1) (reviewed in Woodgett 2005; Franke 2008). Following binding via its PH domain to membrane PIP3, Akt becomes phosphorylated on Thr308 by the membrane-bound PDK1 and on Ser473 by the SIN1-rictor-mTOR complex (Hresko and Mueckler 2005; Sarbassov et al. 2005; Jacinto et al. 2006). Upon activation, Akt phosphorylates numerous downstream targets, both in the cytoplasm and in the nucleus. Through these targets, Akt participates in the regulation of cell survival/apoptosis, cell cycle progression and glucose metabolism (reviewed in Brazil et al. 2004; Manning and Cantley 2007). In neurons, the PI3K/Akt pathway is the major signaling route mediating neuronal survival either by inducing the transcription of survival genes or by inhibiting members of the apoptotic machinery (Kaplan and Miller 2000; Brunet et al. 2001; Fukunaga et al. 2005). Phosphorylation of various neuron-specific targets allows Akt to control different aspects of neuronal function, e.g. GABA receptor stabilization and memory consolidation, dopamine receptor-mediated responses, neuroprotection and neurodegeneration (Brazil et al. 2004; Beaulieu et al. 2007).
In the present study, we explore the possible role of Akt/PKB in regulating cholinergic gene expression in the pheochromocytoma cell line pheochromocytoma 12 (PC12) and in primary septal neurons. By applying several methods of inhibiting Akt activity [a pharmacological inhibitor, an engineered derivative of a regulatory peptide, a dominant-negative (DN) construct and small interfering RNA (siRNA)], we demonstrate that blocking Akt eliminates NGF-evoked cholinergic promoter activity, cholinergic-specific mRNA accumulation, and ACh production. We conclude that Akt plays an essential role in the stimulation of cholinergic gene expression by NGF.
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In this paper, we present data suggesting a novel potential role for the signaling molecule Akt/PKB, specifically in regulating cholinergic differentiation. Using PC12 cells and primary septal neurons, we demonstrated that blocking Akt eliminates NGF-dependent cholinergic promoter activity, ChAT, VAChT, and CHT mRNA accumulation and ACh production. These findings are consistent with our previous reports, based on pharmacological inhibition of PI3K, that showed PI3K-dependent up-regulation of the cholinergic locus and the CHT gene by NGF (Berse et al. 2005; Madziar et al. 2005). Here, we used several parallel methods (pharmacological inhibitors, a DN construct and siRNA) to interfere with either Akt expression or its biological activity. Akt is the major effector of the PI3K pathway, and it is involved in transmission of PI3K survival signals. Therefore, the viability of the treated cells was of concern. We had observed before that extended exposure to the PI3K inhibitor LY294002 in the absence of NGF reduced cell viability (Madziar et al. 2005). However, 3-day treatments with the Akt inhibitors HIMO or TAT-Akt-in did not affect either the general neuronal morphology or viability of primary septal neurons or NGF-differentiated PC12 cells. The cells remained firmly attached to the substratum, their shape and the rate of cell divisions and cell death did not change; they also expressed β-actin at normal levels. In the presence of Akt inhibitors, PC12 cells still formed neurites in response to NGF treatment. Thus, some manifestations of NGF signaling are present even when the activation of Akt is prevented. We demonstrated previously that pharmacological treatments can discriminate between neurite formation and the up-regulation of the cholinergic phenotype in response to NGF, as the former was significantly reduced in PC12 cells treated with MEK inhibitors, while the latter was not. Conversely, inhibition of PI3K with LY294002, which completely blocked the expression of the cholinergic genes, had little effect on neurite formation in PC12 cells (Madziar et al. 2005). The experiments with Akt inhibitors, presented here, confirm that neurite formation in response to NGF is regulated independently from cholinergic differentiation. Taken together, our data suggest that the suppression of cholinergic differentiation in the presence of Akt inhibitors is not the result of a general down-regulation of neuronal properties, but a more specific effect on cholinergic gene expression.
Our results on ChAT and VAChT expression obtained with the PC12 cell line were confirmed in primary cultures obtained from E18 mouse septum. Additionally, the experiments with primary septal cells demonstrated that the expression of another cholinergic marker, CHT, is also regulated by NGF in an Akt-dependent manner. The CHT gene was cloned relatively recently and there is little information available on its regulation. Our data reveal for the first time the specific signals governing the regulation of CHT expression (Berse et al. 2005; Brock et al. 2007; Mellott et al. 2007; this study). The best-described biological function of CHT is providing choline for ACh production. Initial reports, based on the cellular distribution of CHT mRNA, which was found almost exclusively in cholinergic tissues, suggested that CHT is a cholinergic marker co-regulated with ChAT and VAChT (Kobayashi et al. 2002; Ferguson et al. 2003; Kus et al. 2003). Our current data are in agreement with those observations. However, although CHT is essential for the cholinergic phenotype, there are also differences between the regulation of its expression and that of the cholinergic gene locus (Brandon et al. 2004; Berse et al. 2005; Lecomte et al. 2005; Brock et al. 2007). More studies on CHT expression could reveal other potential biological functions of this protein.
The precise mechanism by which the PI3K/Akt pathway regulates cholinergic function is currently unknown. We used FKHRL1 and GSK-3 phosphorylation as indicators of Akt activity, but our experiments did not address the question whether these proteins are the downstream effectors of Akt. However, GSK-3 is a likely and interesting candidate. It is a crucial signaling molecule in several physiological processes and corresponding diseases. It is involved in control of glucose metabolism through insulin actions, type 2 diabetes mellitus (T2DM), apoptosis, Wnt signaling and cancer, and recently it has been linked to Alzheimer’s disease (AD). GSK-3 phosphorylates the protein tau, which leads to neurofibrillary tangles, one of the two key neuropathological features of AD. Also, the anti-apoptotic function of the PI3K/Akt/GSK-3 pathway makes it important for nerve cell survival and our data demonstrate its role in cholinergic function. Thus, a defect in this pathway may contribute to AD in several ways. Furthermore, clinical studies show a higher risk of dementia or significant cognitive decline in diabetic populations, revealing a direct connection between T2DM and AD (for review, see Cole et al. 2007). The results presented here, suggesting that the PI3K/Akt signaling pathway is necessary for the stimulation of ACh synthesis by NGF, are in agreement with those observations. The exact mechanism of insulin resistance in T2DM is not yet known, however the IR seems to function normally in diabetic patients. Thus, it is believed that origin of resistance is downstream from the receptor, and as insulin and NGF share downstream signaling pathways, it can be hypothesized that NGF resistance may also develop in neurons where insulin resistance is present.
Our results with the reporter construct indicate that at least some of the Akt effects on cholinergic gene expression are transcriptional. Akt has been associated mainly with cell survival through interfering with the cytoplasmic mechanisms of apoptosis, but it is also involved in the transcriptional induction of genes related to cell survival, cell cycle, tumor progression, T-cell activation, and energy metabolism (Brunet et al. 2001; Kane and Weiss 2003; Liang and Slingerland 2003; Downward 2004; Agarwal et al. 2005; Huang and Chen 2005; Lee et al. 2005; Porstmann et al. 2005). Some of the transcriptional effects of Akt have been attributed to its inhibitory phosphorylation of GSK-3, which in turn can act as a negative regulator of gene expression (Bijur and Jope 2000). Interestingly, in neuroblastoma cells expressing the estrogen receptor, PI3K/Akt/GSK-3 signaling has been shown to modulate estrogen-dependent gene expression (Mendez and Garcia-Segura 2006). The mechanism of the cross-talk between these pathways is still unclear, although there are indications that it involves regulation of the stability of the estrogen receptor/β-catenin complex by GSK-3. Akt has also been found to regulate gene expression by direct phosphorylation of transcription factors, which leads to their activation (e.g. cAMP-response element binding protein), or inactivation (e.g. the proteins of the Forkhead family) (Hanada et al. 2004). These biological functions of Akt are conducted both in the cytoplasm and in the nucleus. It has been shown recently that the PI3K enhancer (PIKE), a newly identified brain-specific nuclear GTPase, is involved in the anti-apoptotic action of NGF in PC12 cells (Ahn et al. 2004; reviewed in Chan and Ye 2007). It would be interesting to investigate whether nuclear PI3K, nuclear Akt, and/or PI3K enhancer are also involved in the cholinergic differentiation of neurons by NGF.