Molecular interplay between leptin, insulin-like growth factor-1, and β-amyloid in organotypic slices from rabbit hippocampus
© Marwarha et al; licensee BioMed Central Ltd. 2011
Received: 11 April 2011
Accepted: 8 June 2011
Published: 8 June 2011
Evidence shows that the insulin-like growth factor-1 (IGF-1) and leptin reduce β-amyloid (Aβ) production and tau phosphorylation, two major hallmarks of Alzheimer's disease (AD). IGF-1 expression involves the JAK/STAT pathway and the expression of leptin is regulated by the mammalian target of rapamycin complex 1 (mTORC1). We have previously shown that Aβ reduces leptin by inhibiting the mTORC1 pathway and Aβ was also suggested to inhibit the JAK/STAT pathway, potentially attenuating IGF-1 expression. As IGF-1 can activate mTORC1 and leptin can modulate JAK/STAT pathway, we determined the extent to which IGF-1 and leptin can upregulate the expression of one another and protect against Aβ-induced downregulation.
We demonstrate that incubation of organotypic slices from adult rabbit hippocampus with Aβ42 downregulates IGF-1 expression by inhibiting JAK2/STAT5 pathway. Leptin treatment reverses these Aβ42 effects on IGF-1 and treatment with the STAT5 inhibitor completely abrogated the leptin-induced increase in IGF-1. Furthermore, EMSA and ChIP analyses revealed that leptin increases the STAT5 binding to the IGF-1 promoter. We also show that IGF-1 increases the expression of leptin and reverses the Aβ42-induced attenuation in leptin expression via the activation of mTORC1 signaling as the mTORC1 inhibitor rapamycin completely precluded the IGF-1-induced increase in leptin expression.
Our results demonstrate for the first time that Aβ42 downregulates IGF-1 expression and that leptin and IGF-1 rescue one another from downregulation by Aβ42. Our study provides a valuable insight into the leptin/IGF-1/Aβ interplay that may be relevant to the pathophysiology of AD.
KeywordsLeptin IGF-1 Aβ42 mTORC1 C-EBPα STAT5 Organotypic slices
Alzheimer's disease (AD) is pathologically characterized by the deposition and accumulation of β-amyloid (Aβ) peptide in extracellular plaques, the deposition of hyperphosphorylated tau in intracellular neurofibrillary tangles (NFT's), oxidative stress and synaptic loss. Increased levels of Aβ42 (soluble and insoluble) are suggested to play a key role in the neurodegenerative processes that characterize AD. Reduction in the accumulation of this peptide is widely viewed as a potential strategy to protect against AD. There is compelling evidence that the insulin-like growth factor-1 (IGF-1) is involved in the metabolism and clearance of Aβ [1, 2]. Several studies have shown that serum levels of IGF-1 are decreased in AD patients [3–5]. IGF-1 is endogenously produced in the central nervous system [6–8] and is also transported into the brain from the periphery across the blood-brain barrier . In the peripheral system, IGF-1 expression is contingent on the activation of the JAK/STAT pathway, involving the transcription factor STAT5 [10, 11].
Leptin, an adipocytokine produced endogenously in the brain [12–15], has also been shown to reduce Aβ levels in vitro  as well as in vivo [17, 18] and circulating leptin levels are reduced in AD . Expression levels of leptin are regulated by the mammalian target of rapamycin complex 1 (mTORC1) [20–22]. Interestingly, IGF-1 and leptin are interconnected. While IGF-1 activates mTORC1 [23, 24], potentially increasing expression levels of leptin, numerous studies have demonstrated the activation of STAT5 by leptin [25–28] suggesting that leptin may control IGF-1 expression via STAT5 activation. We have recently demonstrated that Aβ42 downregulates leptin expression levels in organotypic hippocampal slices via inhibition of the mTORC1 signaling pathway . However, the extent to which Aβ42 may inhibit IGF-1 expression by inhibiting JAK2/STAT5 has not been determined. Furthermore, the extent to which IGF-1 treatment activates mTORC1 and treatment with leptin activates JAK2/STAT5 respectively precluding Aβ42-induced leptin and IGF-1 downregulation are not known. In this study we found that Aβ42 reduces IGF-1 expression levels by inhibiting JAK2/STAT5 pathway and treatment with leptin prevented these Aβ42 effects. IGF-1 treatment also upregulated leptin levels and prevented Aβ42-induced leptin downregulation by mechanisms involving mTORC1 activation. As increased levels of Aβ42 is a major pathogenic factor in AD, understanding the cellular mechanisms by which IGF-1 and leptin interact to modulate Aβ42 effects may be relevant to the search of agents that preclude the deleterious effects of this peptide.
Aβ42 decreases IGF-1 expression levels and treatment with exogenous leptin reverses the effects of Aβ42
Aβ42 attenuates JAK2/STAT5 signaling and treatment with exogenous leptin restores JAK2/STAT5 signaling
Furthermore, as the nuclear translocation and subsequent transcriptional activity of STAT5 is contingent on phosphorylation, we determined the effect of Aβ42 and leptin treatment on levels of p-Tyr694 STAT5 in the nuclear extracts. We found that Aβ42 treatment completely abolished the translocation of STAT5 to the nucleus, thus mitigating STAT5 transcriptional activity (Figure 2e,f). Leptin treatment, either alone or concomitant with Aβ42, elicited a profound rise in STAT5 translocation to the nucleus (Figure 2e,f).
Leptin induces IGF-1 expression levels via STAT5
Leptin induces IGF-1 expression levels by increasing the binding of STAT5 to the IGF-1 promoter region
IGF-1 increases leptin expression levels and reverses the Aβ42-induced attenuation in leptin expression
IGF-1 increases leptin expression levels via the activation of mTORC1
IGF-1 treatment enhances translation and increases levels of the transcription factor C-EBPα, which mediates increased leptin transcription
This study was conceived to examine the impact of Aβ on the expression of IGF-1 in the hippocampus and assess the role of leptin signaling in the modulation of IGF-1 expression. We demonstrate that Aβ42 induces a marked reduction in IGF-1 expression and treatment with the adipocytokine leptin increases the basal expression levels of IGF-1 and reverses the Aβ42-induced attenuation in IGF-1 expression levels. We further demonstrate that the inhibition of the JAK2/STAT5 underlies Aβ42 and leptin effects on IGF-1 expression, and that IGF-1 expression is mediated by the transcription factor STAT5. We also demonstrate that IGF-1 regulates leptin expression via the mTORC1 signaling pathway by a mechanism that involves the transcription factor C-EBPα. This suggests a mutual positive feedback loop between IGF-1 and leptin and indicates that both IGF-1 and leptin reinforce the expression and activation of each other.
This study demonstrates that Aβ42 inhibits the JAK2/STAT5 pathway. There is evidence that extracellular Aβ is internalized by glial cells via phagocytosis, pinocytosis, and endocytosis [39, 40]. Neurons uptake Aβ from the extracellular milieu as well and this contributes to the accumulation of intraneuronal Aβ . Intraneuronal accumulation of Aβ has been implicated in loss of synaptic plasticity and shown to adversely affect neuronal function and survival [42–44]. Furthermore, it has been demonstrated that intraneuronal Aβ causes memory impairment by attenuating JAK-STAT signaling in hippocampal neurons . IGF-1 expression in the peripheral system is regulated by the transcription factor STAT5 [10, 11, 46]. The functional long-form of leptin receptor (Ob-Rb) is coupled to the JAK2/STAT5 pathway and is highly expressed in the hippocampus [47, 48]. Leptin phosphorylates Ob-Rb at Tyr1138 upon binding and activates the JAK/STAT signal transduction pathway . Leptin binding to Ob-Rb has been shown to activate STAT5 via JAK2 [50–52]. We demonstrate in this study that Aβ42 induces a decrease in p-Tyr1007/1008 JAK2 and p-Tyr694 STAT5 levels, consequently reducing the nuclear translocation of STAT5 and mitigating JAK2/STAT5 signaling. On the other hand, treatment with leptin elicited a significant increase in JAK2/STAT5 activation and reversed the effects of Aβ42 on JAK2/STAT5 signaling, as shown with increased translocation of STAT5 to the nucleus. To determine the extent to which STAT5 mediates leptin effects, we treated organotypic slices with a specific inhibitor of STAT5 in the presence and absence of leptin. We found that STAT5 inhibition markedly reduced IGF-1 expression. As this attenuation of IGF-1 expression by STAT5 inhibition was not alleviated by leptin, such a result suggests that STAT5 is required for leptin-induced increase in IGF-1 expression. We further studied the IGF-1 promoter using EMSA and ChIP analyses to determine the effects of Aβ42 and leptin treatments on IGF-1 transcription and delineate the role of STAT5. We found that Aβ42 reduces the binding of STAT5 in the IGF-1 promoter region. In contrast, both EMSA and ChIP analyses showed that leptin treatment increases STAT5 binding to the IGF-1 promoter region and reverses the attenuating effects of Aβ42 on STAT5 binding in the IGF-1 promoter region. Our data strongly suggest that STAT5 plays an important role in leptin-induced increase in IGF-1 expression.
The findings that Aβ42 reduces IGF-1 expression in the brain and leptin increases the basal levels of this neurotrophic factor and reverses the Aβ-induced decrease in IGF-1 may be of relevance to AD as IGF-1 exhibits neurotrophic, neuromodulatory, neuroendocrine, and metabolic actions in the brain . IGF-1 reduces amyloid burden by increasing its clearance through Aβ carrier proteins like albumin and transthyretin . IGF-1 effects are transduced via the cell surface IGF-1 receptors (IGF1R) belonging to the tyrosine kinase receptor family [54, 55]. The IGF1R are coupled to the PI3K/Akt/mTORC1 pathway . IGF-1 signaling through IGF-1 receptors has been demonstrated to induce the activation of IRS1/PI3K/AkT/mTORC1 pathway and inhibit GSK-3β, thus attenuating tau phosphorylation in NT2N cells  and in primary rat cortical neurons . IGF-1 precludes the β-amyloid-induced neurotoxicity in hippocampal neurons [59, 60] by the activation of PI3K/Akt/mTORC1 pathway . Consistent with this observation, Aβ has been shown to uncouple PI3K/Akt/mTORC1 pathway [61–63]. Furthermore Aβ42 downregulates mTORC1 signaling in SH-SY5Y neuroblastoma cells and mTORC1 signaling is attenuated in APP/PS1 mice model of AD .
We have demonstrated that leptin decreases both basal and Aβ42-induced increase in levels of phosphorylated tau . This study shows that leptin treatment increases IGF-1 expression. We have previously shown that leptin reduces the oxysterol 27-hydroxycholesterol-induced increase in Aβ and phosphorylated tau levels . Several studies have reported the pivotal role of leptin in reducing Aβ production and load [16–18] as well as tau phosphorylation [65, 66]. It is thus conceivable that leptin may, in part, reduce tau phosphorylation by increasing the expression of IGF-1.
Our results demonstrating that IGF-1 regulates leptin suggest that IGF-1 and leptin mutually regulate the expression of each other. We have demonstrated previously that mTORC1 activation is necessary for leptin expression and that the mTORC1 inhibitor rapamycin inhibits leptin expression levels . Furthermore, we demonstrated that Aβ42 inhibits mTORC1 activation and inhibits leptin expression . It is well known that IGF-1 activates the mTORC1 signaling via the Akt signaling pathway [23, 24, 32]. We speculated that IGF-1 may regulate leptin expression through mTORC1 activation and may potentially reverse the deleterious effects of Aβ42 on leptin expression. To this end, we treated organotypic slices with IGF-1 in presence or absence of the mTORC1 inhibitor rapamycin. We found that IGF-1 activates mTORC1 signaling and increases leptin protein and mRNA expression levels. However, in the presence of rapamycin, IGF-1 failed to exert any effect on leptin expression, suggesting that IGF-1 regulates leptin expression via the activation of mTORC1. To determine the effects of IGF-1 treatment on Aβ42-induced downregulation of leptin expression, we incubated organotypic slices with IGF-1 and Aβ42. We found that IGF-1 alleviates the reduction induced by Aβ42 on leptin protein and mRNA expression levels.
Rapamycin is an allosteric inhibitor of mTORC1 that subsequently inhibits translation of proteins that are regulated by mTORC1, including leptin. Although, it is the consensus that rapamycin is a selective inhibitor of mTORC1, recent studies have suggested that under certain conditions, prolonged rapamycin treatment may also inhibit mTORC2 complex [67–69]. mTORC2 was identified as the kinase that activates Akt by phosphorylation at Ser473 . Numerous studies have demonstrated that Akt activates mTORC1 [71, 72]. The fact that mTORC2 phosphorylates Akt at Ser473, and given that Akt activates mTORC1 signaling, indicates that mTORC2 positively regulates mTORC1 signaling. Therefore, inhibition of mTORC2 by rapamycin would result in further indirect inhibition of mTORC1, in addition to the direct allosteric inhibition of mTORC1 by rapamycin . Our results showing that rapamycin also decreases the leptin mRNA levels suggest that mTORC1 is also involved in leptin transcription. To elucidate the role of mTORC1 in the regulation of leptin transcription, we determined the effects of rapamycin on the transcription factors involved in leptin expression. Evidence suggests that the transcription factor C-EBPα plays an indispensable role in leptin expression in the peripheral adipose tissue [35–38]. There are also multiple studies demonstrating the critical role of mTORC1 in the translation of C-EBPα . We found that rapamycin decreases protein levels of C-EBPα in the cytosol as well as in the nucleus. We also determined the involvement of C-EBPα in the Aβ42-induced reduction and IGF-1-induced increase in leptin expression as both Aβ42 and IGF-1 regulate mTORC1 activation and signaling. Western blotting clearly showed that Aβ42 decreases C-EBPα protein levels, while IGF-1 treatment increases the basal levels of C-EBPα and reverses the Aβ42-induced reduction in C-EBPα protein levels. Additionally, ChIP analysis showed that Aβ42 treatment reduces the binding of C-EBPα to the leptin promoter, while treatment with IGF-1 induces an increase in C-EBPα to the leptin promoter.
Leptin, Aβ42, and rapamycin were purchased from Sigma Aldrich (St. Louis, MO). IGF-1 peptide was purchased from Millipore (Bedford, MA). STAT5 inhibitor (573108) was obtained from Calbiochem (San Diego, CA). Hibernate A was obtained from BrainBits LLC (Springfield, IL). Membrane inserts for organotypic slices were from Millipore (Bedford, MA). The antibiotic/antimycotic agents for media (100 U/ml penicillin, and 0.05 μM/ml streptomycin) were purchased from Sigma Aldrich (St. Louis, MO). All other supplies for the culture of organotypic slices (Neurobasal medium, B27, horse serum, and glutamine) were purchased from Invitrogen (Carlsbad, CA).
Organotypic slice preparation and treatment
We chose to use the organotypic slice system for our studies. The organotypic slice system has many advantages in that connectivity between neurons, interneurons and glia is maintained. In addition, we prepared organotypic slices from hippocampus of adult rabbits (2 year-old), a brain region and age that are relevant to the pathophysiology of AD. Additionally, rabbits have a phylogeny closer to humans than rodents , and their Aβ sequence, unlike that of rodents, is similar to the Aβ sequence of the human . Organotypic hippocampal slices were prepared as we have previously shown [14, 15] and as follows. Hippocampi from adult male rabbits (n = 6) were dissected, trimmed of excess white matter and placed into chilled dissection media composed of hibernate A containing 20% horse serum and 0.5 mM l-glutamine. Isolated tissue was placed on a wetted filter paper on the Teflon stage of a MacIlwain chopper for coronal sectioning (300 μm thick). From each rabbit hippocampi, about 50 sections were cut (100 sections per rabbit). Sections were placed in new dissection media and allowed to rest five minutes on ice before separating and plating on membrane inserts. Five sections were placed on each insert with a total of 10 inserts per hippocampus (20 inserts per rabbit). Inserts were placed in 35 mm culture dishes containing 1.1 ml growth media (Neurobasal A with 20% horse serum, 0.5 mM l-glutamine, 100 U/ml penicillin, and 0.05 μM/ml streptomycin), and warmed 30 min prior to plating to ensure complete equilibration. Slices were exposed to a humidified incubator atmosphere (4.5% CO2 and 35°C). Media was changed at 24 h and, at day 4, slices were switched to a defined medium consisting of Neurobasal A, 2% B27 supplement and 0.5 mM l-glutamine. At day 10, organotypic slices from each rabbit were divided into the following treatment groups: (1) vehicle, (2) 125 nM leptin, (3) 80 nM IGF-1, (4) 10 μM Aβ42, (5) 125 nM leptin + 10 μM Aβ42, (6) 80 nM IGF-1 + 10 μM Aβ42, (7) 100 nM rapamycin, (8) 100 nM rapamycin + 80 nM IGF-1, (9) 100 μM STAT5 inhibitor, and (10) 100 μM STAT5 inhibitor + 125 nM leptin. A stock solution of leptin of 62.5 μM (1 mg/ml) was prepared in sterile distilled water and diluted in media at 1:500 to a concentration of 125 nM (2 μg/ml). IGF-1 was procured as a 100 μg lyophilized powder, was dissolved in 1.11 ml sterile distilled water to yield a 12 μM (90 μg/ml) stock solution. The IGF-1 stock solution was further diluted in media at 1:150 to a concentration of 80 nM (600 ng/ml). Aβ42 peptide was dissolved in sterile distilled water to yield a 250 μM (1 mg/ml) stock solution and diluted in media at 1:25 to a final concentration of 10 μM (40 μg/ml). Rapamycin was purchased as a 2.5 mg/ml (2.74 mM) stock solution in DMSO and was diluted in media at 1:274 to yield a working stock solution of 10 μM. The rapamycin solution was further diluted at 1:100 in media to yield a final concentration of 100 nM. Each treatment was delivered into the media of 2 inserts with 5 sections from each of the 6 rabbits. Sections were harvested after 72 h of treatment. The chosen concentrations of leptin (125 nM), Aβ42 (10 μM), and rapamycin (100 nM) were based on our previously published study . The concentration of leptin selected (125 nM) was based on a dose response assay conducted to determine the minimum concentration of leptin that induces phosphorylation of the leptin receptor (Ob-Rb) at Tyr1138 in our organotypic slice paradigm . Other studies have employed 100 nM leptin in SH-SY5Y neuroblastoma cells  and primary neuronal cultures [65, 66]. The rapamycin concentration (100 nM) used was the empirically determined minimum concentration that inhibits mTORC1 activation in our paradigm . Several other studies have utilized up to 1 μM rapamycin to inhibit mTORC1 activation and signaling in SH-SY5Y neuroblastoma cells [64, 75]. The IGF-1 concentration used (80 nM) was empirically determined by a dose response assay with the concentration chosen depicting the minimum concentration that evokes IGF-1 receptor (IGF1R) phosphorylation at Tyr1135/1136 residues in our organotypic slice paradigm. All animal procedures were carried out in accordance with the U.S. Public Health Service Policy on the Humane Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee at the University of North Dakota.
Immunoprecipitation from tissue homogenate was performed for IGF-1 by using "Catch and Release" immunoprecipitation kit from Millipore (Bedford, MA) according to the manufacturer's protocol. Briefly, organotypic slices were homogenized in T-PER tissue protein extraction reagent (Thermo Scientific, Rockford, IL) supplemented with protease and phosphatase inhibitors. Tissue homogenate containing the equivalent to 500 μg of total protein content was incubated with 2 μg of the anti-IGF-1 goat antibody (1:500, Abcam, Cambridge, MA) overnight in the spin columns followed by elution using the denatured elution buffer containing 5% β-mercaptoethanol. 5 μL of the eluate was resolved on a SDS-PAGE gel followed by transfer onto a polyvinylidene difluoride membrane (BioRad, Hercules, CA) and incubation with IGF-1 antibody followed by development with enhanced chemiluminescence (Immun-star HRP chemiluminescent kit, Bio-Rad, Hercules, CA). Bands were visualized on a polyvinylidene difluoride membrane and analyzed by LabWorks 4.5 software on a UVP Bioimaging System (Upland, CA). Quantification of results was performed by densitometry and the results analyzed as total integrated densitometric values (arbitrary units). Rabbit liver tissue homogenate was used as a positive control, while the eluate from the column that did not contain the IGF-1 primary antibody as well as the column that was devoid of the tissue homogenate were used as the negative controls.
Western blot analysis
Organotypic slices were homogenized in NE-PER tissue protein extraction reagent (Thermo Scientific, Rockford, IL) supplemented with protease and phosphatase inhibitors. Protein concentrations from the cytosolic and nuclear homogenates were determined with BCA protein assay. Proteins (10 μg) were separated in SDS-PAGE gels followed by transfer to a polyvinylidene difluoride membrane (BioRad, Hercules, CA) and incubation with the following monoclonal antibodies: anti-JAK2 rabbit antibody (1:1000; Cell Signaling, Boston, MA), anti-phospho (Tyr1007/1008) JAK2 rabbit antibody (1:200; Cell Signaling, Boston, MA), anti-STAT5 rabbit antibody (1:1000; Cell Signaling, Boston, MA), anti-phospho (Tyr694) STAT5 mouse antibody (1:200; Cell Signaling, Boston, MA), anti-IGF1 goat antibody (1:500; Abcam, Cambridge, MA), anti C-EBPα rabbit antibody (Active Motif, Carlsbad, CA). β-actin and lamin A were used as a gel loading control for cytosolic homogenates and nuclear homogenates respectively. The blots were developed with enhanced chemiluminescence (Immun-star HRP chemiluminescent kit, Bio-Rad, Hercules, CA). Bands were visualized on a polyvinylidene difluoride membrane and analyzed by LabWorks 4.5 software on a UVP Bioimaging System (Upland, CA). Quantification of results was performed by densitometry and the results analyzed as total integrated densitometric values (arbitrary units).
Enzyme-linked immunosorbent assay (ELISA)
IGF-1 levels were quantified in the organotypic slices using a quantitative sandwich ELISA kit (R & D systems, Minneapolis, MN) as per the manufacturer's protocol. Organotypic slices were homogenized in T-PER tissue protein extraction reagent (Thermo Scientific, Rockford, IL) supplemented with protease and phosphatase inhibitors. Protein concentrations from tissue homogenates were determined with BCA protein assay. The tissue homogenates belonging to different treatments were further diluted in PBS to yield a protein concentration of 1 mg/ml. 20 μL of the tissue homogenate from each treatment group normalized to 1 mg/ml protein concentration was diluted 1:20 and then further 1:5 in the special buffers provided with the kit to release any IGF-1 that is bound to IGFBP's (IGF-1 binding proteins). A total of 50 μL of this 100-fold diluted homogenate was added to each well of the ELISA plate for the assay. The entire procedure for the assay was performed at 4°C. The optical density of each well was determined using a microplate reader set at 450 nm. The optical density of each well was also determined at 540 nm. The optical density values read at 540 nm were subtracted from the optical density values at 450 nm for each well to account for any optical imperfections of the ELISA plate in accordance with manufacturer's protocol. The concentrations obtained were multiplied by a factor of 100 to account for the 100-fold dilution. The IGF-1 levels were measured in triplicate for each treatment in each of the 6 rabbits. The final results are expressed as ng of IGF-1/ml of tissue homogenate.
Leptin levels were quantified in the organotypic slices using a quantitative sandwich ELISA kit (R & D systems, Minneapolis, MN) as per the manufacturer's protocol. Organotypic slices were homogenized in T-PER tissue protein extraction reagent (Thermo Scientific, Rockford, IL) supplemented with protease and phosphatase inhibitors. Protein concentrations from tissue homogenates were determined with BCA protein assay. The tissue homogenates belonging to different treatments were further diluted in PBS to yield a protein concentration of 1 mg/ml. 1 μL of the tissue homogenate from each treatment group normalized to 1 mg/ml protein concentration was further diluted 1:100 in the assay diluent buffer provided with the kit. A total of 100 μL of this diluted homogenate was added to each well of the ELISA plate for the assay. The optical density of each well was determined using a microplate reader set at 450 nm. The concentrations obtained were multiplied by a factor of 100 to account for the 100-fold dilution. The leptin levels were measured in triplicate for each treatment in each of the 6 rabbits. The final results are expressed as ng of leptin/ml of tissue homogenate.
Quantitative Real time RT-PCR analysis
Primers designed and used for IGF-1, leptin, IGF-1 promoter and leptin promoter
GenBank Accession Number
Site 1 Forward
Site 1 Reverse
Site 2 Forward
Site 2 Reverse
Electrophoretic Mobility Shift Assay (EMSA)
The Electrophoretic Mobility Shift Assay (EMSA) to study the STAT5-IGF-1 promoter interaction was performed using a kit from Active Motif (Carlsbad, CA) following manufacturer's protocol. Nuclear extract was prepared using NE-PER protein extraction reagent following the manufacturer's instructions (Thermo Scientific, Rockford, IL). The human IGF-1 promoter contains two STAT5 binding consensus sequences and these are evolutionary conserved across all mammalian species . The rabbit IGF-1 promoter region spanning 8000 nucleotides upstream of the transcription initiation site in IGF-1 gene was scanned for STAT5 binding consensus sequences using the "TFsearch" online program that searches highly correlated sequence fragments against TFMATRIX transcription factor binding site profile database in 'TRANSFAC' databases [76, 77]. The 5'-biotin labeled and unlabeled oligonucleotide probes that correspond to the STAT5 binding site in the IGF-1 promoter region (Table 1) were purchased from Sigma Aldrich (St Louis, MO). 10 μg of hippocampal nuclear proteins were incubated with either 20 femto moles of biotin labeled oligonucleotide probe or 4 pico moles of unlabelled oligonucleotide. To exhibit specificity of the oligonucleotide probes, unlabelled oligonucleotide probe was used as a specific competitor for binding reactions at a concentration of 200 fold of the concentration of the biotin labeled probe. 1 μg of Poly d(I-C) was used as a non-specific competitor for binding reactions. The resulting binding reaction mix was loaded and resolved on a 5% TBE gel (BioRad, Hercules, CA) followed by transfer onto a nylon membrane. The bands were visualized using the HRP-Streptavidin - Chemiluminescent reaction mix provided with the kit on a UVP Bioimaging System (Upland, CA).
Chromatin Immunoprecipitation (ChIP) Analysis
ChIP analysis was performed to evaluate the extent of STAT5 and C-EBPα binding to the DNA elements in the IGF-1 promoter and leptin promoter regions respectively using "SimpleChIPTM Enzymatic Chromatic IP kit" from Cell Signaling (Boston, MA). Briefly, organotypic slices from each treatment group (~100 mg) were taken and cross-linked with 1% formaldehyde for 15 min followed by the addition of 500 μL of 1.25M glycine solution to cease the cross-linking reaction. The tissue was washed with 4x volumes of 1x PBS and centrifuged at ~220g for 5 min. The pellet was resuspended and incubated for 10 min in 5 ml of tissue lysis buffer containing DTT, protease and phosphatase inhibitors. The subsequent steps to isolate the cross-linked chromatin were performed according to the manufacturer's protocol. One third of the cross-linked chromatin from each sample was set aside as "input" and the rest was subjected to immunoprecipitation. One third of the cross-linked chromatin from each sample was incubated with 5 μg of anti-phospho (Tyr694) STAT5 mouse antibody (Cell Signaling, Boston, MA) or with 5 μg of anti-C-EBPα mouse antibody (Cell Signaling, Boston, MA). One third of the cross-linked chromatin was also incubated with 5 μg of normal Rabbit IgG to serve as negative control. The DNA-protein complexes were collected with Protein G agarose beads and reverse cross-linked by incubation Proteinase K for 2 hours at 65°C followed by elution and purification. The relative abundance of STAT5 binding element in the STAT5 antibody precipitated chromatin and C/EBPα binding element in the C-EBPα antibody precipitated chromatin was determined by qPCR using an iQ SYBR Green Supermix kit following the manufacturer's instructions (BioRad, Hercules, CA) and sequence specific primers (Table 1). The amplification was performed using an iCycler iQ Multicolor Real Time PCR Detection System (BioRad, Hercules, CA). The fold enrichment of the STAT5 binding element and C-EBPα binding element was calculated using the ΔΔCt method  which normalizes ChIP Ct values of each sample to the % input and background.
The significance of differences among the samples was assessed by One Way Analysis of Variance (One Way ANOVA) followed by Tukey's post-hoc test. Statistical analysis was performed with GraphPad Prism software 4.01. Quantitative data for Western blotting analysis are presented as mean values ± S.E.M with unit value assigned to control and the magnitude of differences among the samples being expressed relative to the unit value of control. Quantitative data for ELISA analysis are presented as mean values ± S.E.M with absolute concentrations of IGF-1 and leptin reported. Quantitative data for Real time RT-PCR analysis are presented as mean values ± S.E.M, with reported values being the product of absolute value of the ratio of leptin mRNA to GAPDH mRNA multiplied by 1000000.
Insulin like Growth Factor-1
Janus Kinase 2
Signal Transducer and Activator of Transcription-5
mammalian Target Of Rapamycin Complex 1
mammalian Target Of Rapamycin Complex 2
CCAAT-Enhancer Binding Protein α
This work was supported by a Grant from the NIH (NIEHS, R01ES014826) to OG
- Carro E, Trejo JL, Gomez-Isla T, LeRoith D, Torres-Aleman I: Serum insulin-like growth factor I regulates brain amyloid-beta levels. Nat Med. 2002, 8: 1390-1397.PubMedView ArticleGoogle Scholar
- Hsiao K, Chapman P, Nilsen S, Eckman C, Harigaya Y, Younkin S, Yang F, Cole G: Correlative memory deficits, Abeta elevation, and amyloid plaques in transgenic mice. Science. 1996, 274: 99-102. 10.1126/science.274.5284.99.PubMedView ArticleGoogle Scholar
- Alvarez A, Cacabelos R, Sanpedro C, Garcia-Fantini M, Aleixandre M: Serum TNF-alpha levels are increased and correlate negatively with free IGF-I in Alzheimer disease. Neurobiol Aging. 2007, 28: 533-536. 10.1016/j.neurobiolaging.2006.02.012.PubMedView ArticleGoogle Scholar
- Tei E, Yamamoto H, Watanabe T, Miyazaki A, Nakadate T, Kato N, Mimura M: Use of serum insulin-like growth factor-I levels to predict psychiatric non-response to donepezil in patients with Alzheimer's disease. Growth Horm IGF Res. 2008, 18: 47-54. 10.1016/j.ghir.2007.07.006.PubMedView ArticleGoogle Scholar
- Rivera EJ, Goldin A, Fulmer N, Tavares R, Wands JR, de la Monte SM: Insulin and insulin-like growth factor expression and function deteriorate with progression of Alzheimer's disease: link to brain reductions in acetylcholine. J Alzheimers Dis. 2005, 8: 247-268.PubMedGoogle Scholar
- Rotwein P, Burgess SK, Milbrandt JD, Krause JE: Differential expression of insulin-like growth factor genes in rat central nervous system. Proc Natl Acad Sci USA. 1988, 85: 265-269. 10.1073/pnas.85.1.265.PubMedPubMed CentralView ArticleGoogle Scholar
- Bach MA, Shen-Orr Z, Lowe WL, Roberts CT, LeRoith D: Insulin-like growth factor I mRNA levels are developmentally regulated in specific regions of the rat brain. Brain Res Mol Brain Res. 1991, 10: 43-48.PubMedView ArticleGoogle Scholar
- Bartlett WP, Li XS, Williams M, Benkovic S: Localization of insulin-like growth factor-1 mRNA in murine central nervous system during postnatal development. Dev Biol. 1991, 147: 239-250. 10.1016/S0012-1606(05)80021-1.PubMedView ArticleGoogle Scholar
- Reinhardt RR, Bondy CA: Insulin-like growth factors cross the blood-brain barrier. Endocrinology. 1994, 135: 1753-1761. 10.1210/en.135.5.1753.PubMedGoogle Scholar
- Joung YH, Lee MY, Lim EJ, Kim MS, Hwang TS, Kim SY, Ye SK, Lee JD, Park T, Woo YS, et al: Hypoxia activates the IGF-1 expression through STAT5b in human HepG2 cells. Biochem Biophys Res Commun. 2007, 358: 733-738. 10.1016/j.bbrc.2007.04.201.PubMedView ArticleGoogle Scholar
- Li L, He D, Wilborn TW, Falany JL, Falany CN: Increased SULT1E1 activity in HepG2 hepatocytes decreases growth hormone stimulation of STAT5b phosphorylation. Steroids. 2009, 74: 20-29. 10.1016/j.steroids.2008.09.002.PubMedPubMed CentralView ArticleGoogle Scholar
- Li HY, Wang LL, Yeh RS: Leptin immunoreactivity in the central nervous system in normal and diabetic rats. Neuroreport. 1999, 10: 437-442. 10.1097/00001756-199902050-00042.PubMedView ArticleGoogle Scholar
- Ur E, Wilkinson DA, Morash BA, Wilkinson M: Leptin immunoreactivity is localized to neurons in rat brain. Neuroendocrinology. 2002, 75: 264-272. 10.1159/000054718.PubMedView ArticleGoogle Scholar
- Marwarha G, Dasari B, Prasanthi JR, Schommer J, Ghribi O: Leptin reduces the accumulation of Abeta and phosphorylated tau induced by 27-hydroxycholesterol in rabbit organotypic slices. J Alzheimers Dis. 2010, 19: 1007-1019.PubMedPubMed CentralGoogle Scholar
- Marwarha G, Dasari B, Prabhakara JP, Schommer J, Ghribi O: beta-Amyloid regulates leptin expression and tau phosphorylation through the mTORC1 signaling pathway. J Neurochem. 2010, 115: 373-384. 10.1111/j.1471-4159.2010.06929.x.PubMedPubMed CentralView ArticleGoogle Scholar
- Greco SJ, Sarkar S, Johnston JM, Tezapsidis N: Leptin regulates tau phosphorylation and amyloid through AMPK in neuronal cells. Biochem Biophys Res Commun. 2009, 380: 98-104. 10.1016/j.bbrc.2009.01.041.PubMedPubMed CentralView ArticleGoogle Scholar
- Fewlass DC, Noboa K, Pi-Sunyer FX, Johnston JM, Yan SD, Tezapsidis N: Obesity-related leptin regulates Alzheimer's Abeta. FASEB J. 2004, 18: 1870-1878. 10.1096/fj.04-2572com.PubMedView ArticleGoogle Scholar
- Tezapsidis N, Johnston JM, Smith MA, Ashford JW, Casadesus G, Robakis NK, Wolozin B, Perry G, Zhu X, Greco SJ, et al: Leptin: a novel therapeutic strategy for Alzheimer's disease. J Alzheimers Dis. 2009, 16: 731-740.PubMedPubMed CentralGoogle Scholar
- Power DA, Noel J, Collins R, O'Neill D: Circulating leptin levels and weight loss in Alzheimer's disease patients. Dement Geriatr Cogn Disord. 2001, 12: 167-170. 10.1159/000051252.PubMedView ArticleGoogle Scholar
- Roh C, Han J, Tzatsos A, Kandror KV: Nutrient-sensing mTOR-mediated pathway regulates leptin production in isolated rat adipocytes. Am J Physiol Endocrinol Metab. 2003, 284: E322-E330.PubMedView ArticleGoogle Scholar
- Cho HJ, Park J, Lee HW, Lee YS, Kim JB: Regulation of adipocyte differentiation and insulin action with rapamycin. Biochem Biophys Res Commun. 2004, 321: 942-948. 10.1016/j.bbrc.2004.07.050.PubMedView ArticleGoogle Scholar
- Chakrabarti P, Anno T, Manning BD, Luo Z, Kandror KV: The mammalian target of rapamycin complex 1 regulates leptin biosynthesis in adipocytes at the level of translation: the role of the 5'-untranslated region in the expression of leptin messenger ribonucleic acid. Mol Endocrinol. 2008, 22: 2260-2267. 10.1210/me.2008-0148.PubMedPubMed CentralView ArticleGoogle Scholar
- Vivanco I, Sawyers CL: The phosphatidylinositol 3-Kinase AKT pathway in human cancer. Nat Rev Cancer. 2002, 2: 489-501. 10.1038/nrc839.PubMedView ArticleGoogle Scholar
- Grimberg A: Mechanisms by which IGF-I may promote cancer. Cancer Biol Ther. 2003, 2: 630-635.PubMedPubMed CentralView ArticleGoogle Scholar
- Seufert J, Kieffer TJ, Habener JF: Leptin inhibits insulin gene transcription and reverses hyperinsulinemia in leptin-deficient ob/ob mice. Proc Natl Acad Sci USA. 1999, 96: 674-679. 10.1073/pnas.96.2.674.PubMedPubMed CentralView ArticleGoogle Scholar
- Carvalheira JB, Ribeiro EB, Folli F, Velloso LA, Saad MJ: Interaction between leptin and insulin signaling pathways differentially affects JAK-STAT and PI 3-kinase-mediated signaling in rat liver. Biol Chem. 2003, 384: 151-159. 10.1515/BC.2003.016.PubMedView ArticleGoogle Scholar
- Laubner K, Kieffer TJ, Lam NT, Niu X, Jakob F, Seufert J: Inhibition of preproinsulin gene expression by leptin induction of suppressor of cytokine signaling 3 in pancreatic beta-cells. Diabetes. 2005, 54: 3410-3417. 10.2337/diabetes.54.12.3410.PubMedView ArticleGoogle Scholar
- Gong Y, Ishida-Takahashi R, Villanueva EC, Fingar DC, Munzberg H, Myers MG: The long form of the leptin receptor regulates STAT5 and ribosomal protein S6 via alternate mechanisms. J Biol Chem. 2007, 282: 31019-31027. 10.1074/jbc.M702838200.PubMedView ArticleGoogle Scholar
- Muller J, Sperl B, Reindl W, Kiessling A, Berg T: Discovery of chromone-based inhibitors of the transcription factor STAT5. Chembiochem. 2008, 9: 723-727. 10.1002/cbic.200700701.PubMedView ArticleGoogle Scholar
- Wang Y, Jiang H: Identification of a distal STAT5-binding DNA region that may mediate growth hormone regulation of insulin-like growth factor-I gene expression. J Biol Chem. 2005, 280: 10955-10963. 10.1074/jbc.M412808200.PubMedView ArticleGoogle Scholar
- Eleswarapu S, Gu Z, Jiang H: Growth hormone regulation of insulin-like growth factor-I gene expression may be mediated by multiple distal signal transducer and activator of transcription 5 binding sites. Endocrinology. 2008, 149: 2230-2240. 10.1210/en.2007-1344.PubMedPubMed CentralView ArticleGoogle Scholar
- Giorgetti S, Ballotti R, Kowalski-Chauvel A, Tartare S, Van OE: The insulin and insulin-like growth factor-I receptor substrate IRS-1 associates with and activates phosphatidylinositol 3-kinase in vitro. J Biol Chem. 1993, 268: 7358-7364.PubMedGoogle Scholar
- Burgos SA, Cant JP: IGF-1 stimulates protein synthesis by enhanced signaling through mTORC1 in bovine mammary epithelial cells. Domest Anim Endocrinol. 2010, 38: 211-221. 10.1016/j.domaniend.2009.10.005.PubMedView ArticleGoogle Scholar
- Calkhoven CF, Muller C, Leutz A: Translational control of C/EBPalpha and C/EBPbeta isoform expression. Genes Dev. 2000, 14: 1920-1932.PubMedPubMed CentralGoogle Scholar
- Hwang CS, Loftus TM, Mandrup S, Lane MD: Adipocyte differentiation and leptin expression. Annu Rev Cell Dev Biol. 1997, 13: 231-259. 10.1146/annurev.cellbio.13.1.231.PubMedView ArticleGoogle Scholar
- Mason MM, He Y, Chen H, Quon MJ, Reitman M: Regulation of leptin promoter function by Sp1, C/EBP, and a novel factor. Endocrinology. 1998, 139: 1013-1022. 10.1210/en.139.3.1013.PubMedGoogle Scholar
- Krempler F, Breban D, Oberkofler H, Esterbauer H, Hell E, Paulweber B, Patsch W: Leptin, peroxisome proliferator-activated receptor-gamma, and CCAAT/enhancer binding protein-alpha mRNA expression in adipose tissue of humans and their relation to cardiovascular risk factors. Arterioscler Thromb Vasc Biol. 2000, 20: 443-449.PubMedView ArticleGoogle Scholar
- Ramji DP, Foka P: CCAAT/enhancer-binding proteins: structure, function and regulation. Biochem J. 2002, 365: 561-575.PubMedPubMed CentralView ArticleGoogle Scholar
- Matsunaga W, Shirokawa T, Isobe K: Specific uptake of Abeta1-40 in rat brain occurs in astrocyte, but not in microglia. Neurosci Lett. 2003, 342: 129-131. 10.1016/S0304-3940(03)00240-4.PubMedView ArticleGoogle Scholar
- Mandrekar S, Jiang Q, Lee CY, Koenigsknecht-Talboo J, Holtzman DM, Landreth GE: Microglia mediate the clearance of soluble Abeta through fluid phase macropinocytosis. J Neurosci. 2009, 29: 4252-4262. 10.1523/JNEUROSCI.5572-08.2009.PubMedPubMed CentralView ArticleGoogle Scholar
- LaFerla FM, Green KN, Oddo S: Intracellular amyloid-beta in Alzheimer's disease. Nat Rev Neurosci. 2007, 8: 499-509. 10.1038/nrn2168.PubMedView ArticleGoogle Scholar
- Chui DH, Dobo E, Makifuchi T, Akiyama H, Kawakatsu S, Petit A, Checler F, Araki W, Takahashi K, Tabira T: Apoptotic neurons in Alzheimer's disease frequently show intracellular Abeta42 labeling. J Alzheimers Dis. 2001, 3: 231-239.PubMedGoogle Scholar
- Oddo S, Caccamo A, Shepherd JD, Murphy MP, Golde TE, Kayed R, Metherate R, Mattson MP, Akbari Y, LaFerla FM: Triple-transgenic model of Alzheimer's disease with plaques and tangles: intracellular Abeta and synaptic dysfunction. Neuron. 2003, 39: 409-421. 10.1016/S0896-6273(03)00434-3.PubMedView ArticleGoogle Scholar
- Billings LM, Oddo S, Green KN, McGaugh JL, LaFerla FM: Intraneuronal Abeta causes the onset of early Alzheimer's disease-related cognitive deficits in transgenic mice. Neuron. 2005, 45: 675-688. 10.1016/j.neuron.2005.01.040.PubMedView ArticleGoogle Scholar
- Chiba T, Yamada M, Sasabe J, Terashita K, Shimoda M, Matsuoka M, Aiso S: Amyloid-beta causes memory impairment by disturbing the JAK2/STAT3 axis in hippocampal neurons. Mol Psychiatry. 2009, 14: 206-222. 10.1038/mp.2008.105.PubMedView ArticleGoogle Scholar
- Roberts TK, Bailey JL: Beyond nutrition: neuropeptide signaling and muscle mass maintenance in chronic kidney disease. Kidney Int. 2008, 74: 143-145. 10.1038/ki.2008.220.PubMedView ArticleGoogle Scholar
- Mercer JG, Hoggard N, Williams LM, Lawrence CB, Hannah LT, Trayhurn P: Localization of leptin receptor mRNA and the long form splice variant (Ob-Rb) in mouse hypothalamus and adjacent brain regions by in situ hybridization. FEBS Lett. 1996, 387: 113-116. 10.1016/0014-5793(96)00473-5.PubMedView ArticleGoogle Scholar
- Shanley LJ, O'Malley D, Irving AJ, Ashford ML, Harvey J: Leptin inhibits epileptiform-like activity in rat hippocampal neurones via PI 3-kinase-driven activation of BK channels. J Physiol. 2002, 545: 933-944. 10.1113/jphysiol.2002.029488.PubMedPubMed CentralView ArticleGoogle Scholar
- Bahrenberg G, Behrmann I, Barthel A, Hekerman P, Heinrich PC, Joost HG, Becker W: Identification of the critical sequence elements in the cytoplasmic domain of leptin receptor isoforms required for Janus kinase/signal transducer and activator of transcription activation by receptor heterodimers. Mol Endocrinol. 2002, 16: 859-872. 10.1210/me.16.4.859.PubMedView ArticleGoogle Scholar
- Ghilardi N, Ziegler S, Wiestner A, Stoffel R, Heim MH, Skoda RC: Defective STAT signaling by the leptin receptor in diabetic mice. Proc Natl Acad Sci USA. 1996, 93: 6231-6235. 10.1073/pnas.93.13.6231.PubMedPubMed CentralView ArticleGoogle Scholar
- Ghilardi N, Skoda RC: The leptin receptor activates janus kinase 2 and signals for proliferation in a factor-dependent cell line. Mol Endocrinol. 1997, 11: 393-399. 10.1210/me.11.4.393.PubMedView ArticleGoogle Scholar
- Morton NM, Emilsson V, Liu YL, Cawthorne MA: Leptin action in intestinal cells. J Biol Chem. 1998, 273: 26194-26201. 10.1074/jbc.273.40.26194.PubMedView ArticleGoogle Scholar
- Torres-Aleman I: Serum growth factors and neuroprotective surveillance: focus on IGF-1. Mol Neurobiol. 2000, 21: 153-160. 10.1385/MN:21:3:153.PubMedView ArticleGoogle Scholar
- Ullrich A, Bell JR, Chen EY, Herrera R, Petruzzelli LM, Dull TJ, Gray A, Coussens L, Liao YC, Tsubokawa M, et al: Human insulin receptor and its relationship to the tyrosine kinase family of oncogenes. Nature. 1985, 313: 756-761. 10.1038/313756a0.PubMedView ArticleGoogle Scholar
- LeRoith D, Werner H, Faria TN, Kato H, Adamo M, Roberts CT: Insulin-like growth factor receptors. Implications for nervous system function. Ann N Y Acad Sci. 1993, 692: 22-32. 10.1111/j.1749-6632.1993.tb26202.x.PubMedView ArticleGoogle Scholar
- Zheng WH, Kar S, Dore S, Quirion R: Insulin-like growth factor-1 (IGF-1): a neuroprotective trophic factor acting via the Akt kinase pathway. J Neural Transm Suppl. 2000, 261-272.Google Scholar
- Hong M, Lee VM: Insulin and insulin-like growth factor-1 regulate tau phosphorylation in cultured human neurons. J Biol Chem. 1997, 272: 19547-19553. 10.1074/jbc.272.31.19547.PubMedView ArticleGoogle Scholar
- Lesort M, Johnson GV: Insulin-like growth factor-1 and insulin mediate transient site-selective increases in tau phosphorylation in primary cortical neurons. Neuroscience. 2000, 99: 305-316. 10.1016/S0306-4522(00)00200-1.PubMedView ArticleGoogle Scholar
- Dore S, Kar S, Quirion R: Insulin-like growth factor I protects and rescues hippocampal neurons against beta-amyloid- and human amylin-induced toxicity. Proc Natl Acad Sci USA. 1997, 94: 4772-4777. 10.1073/pnas.94.9.4772.PubMedPubMed CentralView ArticleGoogle Scholar
- Dore S, Bastianetto S, Kar S, Quirion R: Protective and rescuing abilities of IGF-I and some putative free radical scavengers against beta-amyloid-inducing toxicity in neurons. Ann N Y Acad Sci. 1999, 890: 356-364. 10.1111/j.1749-6632.1999.tb08015.x.PubMedView ArticleGoogle Scholar
- Nassif M, Hoppe J, Santin K, Frozza R, Zamin LL, Simao F, Horn AP, Salbego C: Beta-amyloid peptide toxicity in organotypic hippocampal slice culture involves Akt/PKB, GSK-3beta, and PTEN. Neurochem Int. 2007, 50: 229-235. 10.1016/j.neuint.2006.08.008.PubMedView ArticleGoogle Scholar
- Chen TJ, Wang DC, Chen SS: Amyloid-beta interrupts the PI3K-Akt-mTOR signaling pathway that could be involved in brain-derived neurotrophic factor-induced Arc expression in rat cortical neurons. J Neurosci Res. 2009, 87: 2297-2307. 10.1002/jnr.22057.PubMedView ArticleGoogle Scholar
- Lee HK, Kumar P, Fu Q, Rosen KM, Querfurth HW: The insulin/Akt signaling pathway is targeted by intracellular beta-amyloid. Mol Biol Cell. 2009, 20: 1533-1544. 10.1091/mbc.E08-07-0777.PubMedPubMed CentralView ArticleGoogle Scholar
- Lafay-Chebassier C, Paccalin M, Page G, Barc-Pain S, Perault-Pochat MC, Gil R, Pradier L, Hugon J: mTOR/p70S6k signalling alteration by Abeta exposure as well as in APP-PS1 transgenic models and in patients with Alzheimer's disease. J Neurochem. 2005, 94: 215-225. 10.1111/j.1471-4159.2005.03187.x.PubMedView ArticleGoogle Scholar
- Greco SJ, Sarkar S, Johnston JM, Zhu X, Su B, Casadesus G, Ashford JW, Smith MA, Tezapsidis N: Leptin reduces Alzheimer's disease-related tau phosphorylation in neuronal cells. Biochem Biophys Res Commun. 2008, 376: 536-541. 10.1016/j.bbrc.2008.09.026.PubMedPubMed CentralView ArticleGoogle Scholar
- Greco SJ, Sarkar S, Casadesus G, Zhu X, Smith MA, Ashford JW, Johnston JM, Tezapsidis N: Leptin inhibits glycogen synthase kinase-3beta to prevent tau phosphorylation in neuronal cells. Neurosci Lett. 2009, 455: 191-194. 10.1016/j.neulet.2009.03.066. 5PubMedPubMed CentralView ArticleGoogle Scholar
- Sarbassov DD, Ali SM, Sengupta S, Sheen JH, Hsu PP, Bagley AF, Markhard AL, Sabatini DM: Prolonged rapamycin treatment inhibits mTORC2 assembly and Akt/PKB. Mol Cell. 2006, 22: 159-168. 10.1016/j.molcel.2006.03.029.PubMedView ArticleGoogle Scholar
- Zeng Z, Sarbassov dD, Samudio IJ, Yee KW, Munsell MF, Ellen JC, Giles FJ, Sabatini DM, Andreeff M, Konopleva M: Rapamycin derivatives reduce mTORC2 signaling and inhibit AKT activation in AML. Blood. 2007, 109: 3509-3512. 10.1182/blood-2006-06-030833.PubMedPubMed CentralView ArticleGoogle Scholar
- Barilli A, Visigalli R, Sala R, Gazzola GC, Parolari A, Tremoli E, Bonomini S, Simon A, Closs EI, Dall'Asta V, et al: In human endothelial cells rapamycin causes mTORC2 inhibition and impairs cell viability and function. Cardiovasc Res. 2008, 78: 563-571. 10.1093/cvr/cvn024.PubMedView ArticleGoogle Scholar
- Sarbassov DD, Guertin DA, Ali SM, Sabatini DM: Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex. Science. 2005, 307: 1098-1101. 10.1126/science.1106148.PubMedView ArticleGoogle Scholar
- Brown EJ, Beal PA, Keith CT, Chen J, Shin TB, Schreiber SL: Control of p70 s6 kinase by kinase activity of FRAP in vivo. Nature. 1995, 377: 441-446. 10.1038/377441a0.PubMedView ArticleGoogle Scholar
- Peterson RT, Beal PA, Comb MJ, Schreiber SL: FKBP12-rapamycin-associated protein (FRAP) autophosphorylates at serine 2481 under translationally repressive conditions. J Biol Chem. 2000, 275: 7416-7423. 10.1074/jbc.275.10.7416.PubMedView ArticleGoogle Scholar
- Graur D, Duret L, Gouy M: Phylogenetic position of the order Lagomorpha (rabbits, hares and allies). Nature. 1996, 379: 333-335. 10.1038/379333a0.PubMedView ArticleGoogle Scholar
- Johnstone EM, Chaney MO, Norris FH, Pascual R, Little SP: Conservation of the sequence of the Alzheimer's disease amyloid peptide in dog, polar bear and five other mammals by cross-species polymerase chain reaction analysis. Brain Res Mol Brain Res. 1991, 10: 299-305.PubMedView ArticleGoogle Scholar
- Lafay-Chebassier C, Perault-Pochat MC, Page G, Rioux BA, Damjanac M, Pain S, Houeto JL, Gil R, Hugon J: The immunosuppressant rapamycin exacerbates neurotoxicity of Abeta peptide. J Neurosci Res. 2006, 84: 1323-1334. 10.1002/jnr.21039.PubMedView ArticleGoogle Scholar
- Wingender E, Kel AE, Kel OV, Karas H, Heinemeyer T, Dietze P, Knuppel R, Romaschenko AG, Kolchanov NA: TRANSFAC, TRRD and COMPEL: towards a federated database system on transcriptional regulation. Nucleic Acids Res. 1997, 25: 265-268. 10.1093/nar/25.1.265.PubMedPubMed CentralView ArticleGoogle Scholar
- Heinemeyer T, Wingender E, Reuter I, Hermjakob H, Kel AE, Kel OV, Ignatieva EV, Ananko EA, Podkolodnaya OA, Kolpakov FA, et al: Databases on transcriptional regulation: TRANSFAC, TRRD and COMPEL. Nucleic Acids Res. 1998, 26: 362-367. 10.1093/nar/26.1.362.PubMedPubMed CentralView ArticleGoogle Scholar
- Livak KJ, Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001, 25: 402-408. 10.1006/meth.2001.1262.PubMedView ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.