Effects of ketamine administration on mTOR and reticulum stress signaling pathways in the brain after the infusion of rapamycin into prefrontal cortex

Recent studies show that activation of the mTOR signaling pathway is required for the rapid antidepressant actions of glutamate N-methyl-D-aspartate (NMDA) receptor antagonists. A relationship between mTOR kinase and the endoplasmic reticulum (ER) stress pathway, also known as the unfolded protein response (UPR) has been shown. We evaluate the effects of ketamine administration on the mTOR signaling pathway and proteins of UPR in the prefrontal cortex (PFC), hippocampus, amygdala and nucleus accumbens, after the inhibiton of mTOR signaling in the PFC. Male adult Wistar rats received pharmacological mTOR inhibitor, rapamycin (0.2 nmol), or vehicle into the PFC and then a single dose of ketamine (15 mg/kg, i.p.). The immunocontent of mTOR, eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), eukaryotic elongation factor 2 kinase (eEF2K) homologous protein (CHOP), PKR-like ER kinase (PERK) and inositol-requiring enzyme 1 (IRE1) – alpha were determined in the brain. The mTOR levels were reduced in the rapamycin group treated with saline and ketamine in the PFC; p4EBP1 levels were reduced in the rapamycin group treated with ketamine in the PFC and nucleus accumbens; the levels of peEF2K were increased in the PFC in the vehicle group treated with ketamine and reduced in the rapamycin group treated with ketamine. The PERK and IRE1-alpha levels were decreased in the PFC in the rapamycin group treated with ketamine. Our results suggest that mTOR signaling inhibition by rapamycin could be involved, at least in part, with the mechanism of action of ketamine; and the ketamine antidepressant on ER stress pathway could be also mediated by mTOR signaling pathway in certain brain structures.     

Alterations in mammalian target of rapamycin signaling pathways after traumatic brain injury.

In response to traumatic brain injury (TBI), neurons initiate neuroplastic processes through the activation of intracellular signaling pathways. However, the molecular mechanisms underlying neuroplasticity after TBI are poorly understood. To study this, we utilized the fluid-percussion brain injury (FPI) model to investigate alterations in the mammalian target of rapamycin (mTOR) signaling pathways in response to TBI. Mammalian target of rapamycin stimulates mRNA translation through phosphorylation of eukaryotic initiation factor 4E binding protein-1 (4E-BP1), p70 ribosomal S6 kinase (p70S6K), and ribosomal protein S6 (rpS6). These pathways coordinate cell growth and neuroplasticity via dendritic protein synthesis. Rats received sham surgery or moderate parasagittal FPI on the right side of the parietal cortex, followed by 15 mins, 30 mins, 4 h, 24 h, or 72 h of recovery. Using Western blot analysis, we found that mTOR, p70S6K, rpS6, and 4E-BP1 phosphorylation levels were significantly increased in the ipsilateral parietal cortex and hippocampus from 30 mins to 24 h after TBI, whereas total protein levels were unchanged. Using confocal microscopy to localize these changes, we found that rpS6 phosphorylation was increased in the parietal cortex and all subregions of the hippocampus. In accordance with these results, eIF4E, a key, rate-limiting mRNA translation factor, was also phosphorylated by mitogen-activated protein kinase-interacting kinase 1 (Mnk1) 15 mins after TBI. Together, these results suggest that changes in mRNA translation may be one mechanism that neurons use to respond to trauma and may contribute to the neuroplastic changes observed after TBI.     

Sirt1 overexpression in neurons promotes neurite outgrowth and cell survival through inhibition of the mTOR signaling

The mammalian nicotinamide-adenine dinucleotide (NAD)-dependent deacetylase Sirt1 impacts different processes involved in the maintenance of brain integrity and in the pathogenic pathways associated with several neurodegenerative disorders, including Alzheimer's disease. Here we used human Sirt1 transgenic mice to demonstrate that neuron-specific Sirt1 overexpression promoted neurite outgrowth and improved cell viability under normal and nutrient-limiting conditions in primary culture systems and that Sirt1-overexpressing neurons exhibited higher tolerance to cell death or degeneration induced by amyloid-β1-42 oligomers. Coincidentally, we found that enhanced Sirt1 expression in neurons downregulated the mammalian target of rapamycin (mTOR) protein levels and its phosphorylation without changes in its mRNA levels, which was accompanied by concomitant inhibition of the mTOR downstream signaling activity as revealed by decreased p70S6 kinase (p70S6K) phosphorylation at Thr389. Consistently with this, using a Sirt1 siRNA transfection approach, we observed that reduction of endogenous mouse Sirt1 led to increased levels of mTOR and phosphorylation of itself and p70S6K as well as impaired cell survival and neurite outgrowth in wild-type mouse primary neurons, corroborating a suppressing effect of mTOR by Sirt1. Correspondingly, the mTOR inhibitor rapamycin markedly improved neuronal cell survival in response to nutrient deprivation and significantly enhanced neurite outgrowth in wild-type mouse neurons. The protective effect of rapamycin was extended to neurons even with Sirt1 siRNA knockdown that displayed developmental abnormalities compared with siRNA control-treated cells. Collectively, our findings suggest that Sirt1 may act to promote growth and survival of neurons in the central nervous system via its negative modulation of mTOR signaling.     

Activation of mammalian target of rapamycin contributes to pain nociception induced in rats by BmK I,a sodium channel-specific modulator

The mammalian target of rapamycin (mTOR) pathway is essential for maintenance of the sensitivity of certain adult sensory neurons. Here, we investigated whether the mTOR cascade is involved in scorpion envenomation-induced pain hypersensitivity in rats. The results showed that intraplantar injection of a neurotoxin from Buthus martensii Karsch, BmK I (10 μg), induced the activation of mTOR, as well as its downstream molecules p70 ribosomal S6 protein kinase (p70 S6K) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), in lumbar 5–6 dorsal root ganglia neurons on both sides in rats. The activation peaked at 2 h and recovered 1 day after injection. Compared with the control group, the ratios of p-mTOR/p-p70 S6K/p-4EBP1 in three types of neurons changed significantly. The cell typology of p-mTOR/p-p70 S6K/p-4E-BP1 immuno-reactive neurons also changed. Intrathecal administration of deforolimus, a specific inhibitor of mTOR, attenuated BmK I-induced pain responses (spontaneous flinching, paroxysmal pain-like behavior, and mechanical hypersensitivity). Together, these results imply that the mTOR signaling pathway is mobilized by and contributes to experimental scorpion sting-induced pain.     

Chronic alcohol consumption alters mammalian target of rapamycin (mTOR), reduces ribosomal p70s6 kinase and p4E-BP1 levels in mouse cerebral cortex

Reduced insulin sensitivity following chronic alcohol consumption may contribute to alcohol-induced brain damage although the underlying mechanism(s) has not been elucidated. This study was designed to examine the effect of chronic alcohol intake on insulin signaling in mouse cerebral cortex. FVB mice were fed with a 4% alcohol diet for 16 weeks. Insulin receptor substrates (IRS-1, IRS-2) and post-receptor signaling molecules Akt, mammalian target of rapamycin (mTOR), ribosomal p70s6 kinase (p70s6k) and the eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1) as well as the apoptotic marker caspase-3 were evaluated using Western blot analysis. Chronic alcohol intake significantly dampened whole body glucose tolerance, enhanced expression of caspase-3 and mTOR, reduced p70s6k and 4E-BP1 with little effect on Akt signaling in alcohol-consuming mice. These data suggest that chronic alcohol intake may contribute to cerebral cortex dysfunction through mechanisms related, at least in part, to dampened post insulin receptor signaling at the levels of mTOR, p70s6k and 4E-BP1.     

Early molecular and behavioral response to lipopolysaccharide in the WAG/Rij rat model of absence epilepsy and depressive-like behavior,involves interplay between AMPK,AKT/mTOR pathways and neuroinflammatory cytokine release

The mammalian target of rapamycin (mTOR) pathway has been recently indicated as a suitable drug target for the prevention of epileptogenesis. The mTOR pathway is known for its involvement in the control of the immune system. Since neuroinflammation is recognized as a major contributor to epileptogenesis, we wished to examine whether the neuroprotective effects of mTOR modulation could involve a suppression of the neuroinflammatory process in epileptic brain. We have investigated the early molecular mechanisms involved in the effects of intracerebral administration of the lipopolysaccharide (LPS) in the WAG/Rij rat model of absence epilepsy, in relation to seizure generation and depressive-like behavior; we also tested whether the effects of LPS could be modulated by treatment with rapamycin (RAP), a specific mTOR inhibitor. We determined, in specific rat brain areas, levels of p-mTOR/p-p70S6K and also p-AKT/p-AMPK as downstream or upstream indicators of mTOR activity and tested the effects of LPS and RAP co-administration. Changes in the brain levels of pro-inflammatory cytokines IL-1β and TNF-α and their relative mRNA expression levels were measured, and the involvement of nuclear factor-κB (NF-κB) was also examined in vitro. We confirmed that RAP inhibits the aggravation of absence seizures and depressive-like/sickness behavior induced by LPS in the WAG/Rij rats through the activation of mTOR and show that this effect is correlated with the ability of RAP to dampen and delay LPS increases in neuroinflammatory cytokines IL-1β and TNF-α, most likely through inhibition of the activation of NF-κB. Our results suggest that such a mechanism could contribute to the antiseizure, antiepileptogenic and behavioral effects of RAP and further highlight the potential therapeutic usefulness of mTOR inhibition in the management of human epilepsy and other neurological disorders. Furthermore, we show that LPS-dependent neuroinflammatory effects are also mediated by a complex interplay between AKT, AMPK and mTOR with specificity to selective brain areas. In conclusion, neuroinflammation appears to be a highly coordinated phenomenon, where timing of intervention may be carefully evaluated in order to identify the best suitable target.     

Dissociation of Akt/PKB and ribosomal S6 kinase signaling markers in a transgenic mouse model of Alzheimer's disease

Previous studies demonstrated that the PKR (double-stranded RNA-activated protein kinase) pathway was activated while the mTOR (mammalian target of rapamycin) pathway was inhibited in Alzheimer's disease (AD). Here, we analysed upstream and downstream factors of mTOR in brain of APP(SL)/PS1 KI mice displaying a massive neuronal loss in hippocampus. While mTOR levels were not modified, we found a great activation of Akt with a robust accumulation of P-Akt((T308)) in non-apoptotic neurons at 6 months of age. At the opposite, a significant decrease of the p70/85S6K activation was observed in brain of PS1 KI and APP(SL)/PS1 KI mice with a very weak or no nucleocytoplasmic P-p70/85S6K((T389)) staining in apoptotic neurons of APP(SL)/PS1 KI mice. Furthermore, the activation of Erk1/2, 4E-BP1 and p70S6K((T421/S424)) (substrate of Erk1/2), except eIF4E, was not modified. These findings demonstrate a clear dissociation between Akt and ribosomal S6K signaling markers in these mice which could be involved in the AD pathological process.     

mTOR inhibitor rapamycin suppresses striatal post-ischemic LTP

The two complexes of the mammalian target of rapamycin (mTOR), mTORC1 and mTORC2, have central functions in the integration of both extracellular and intracellular signals that are also critical players in the induction of post-ischemic long-term potentiation (i-LTP), a pathological form of plasticity inducible in striatal medium spiny neurons (MSNs) after a brief episode of in vitro ischemia. To evaluate the involvement of mTOR complexes during ischemia we analyzed the time course of i-LTP by intracellular recordings of MSNs from corticostriatal slices incubated with 1 μM mTOR inhibitor rapamycin. Although rapamycin did not affect the amplitude and duration of ischemia-induced membrane depolarization it fully prevented i-LTP, leaving unaffected the capability to undergo activity-dependent LTP following high-frequency stimulation of corticostriatal fibers. The present results argue for a role of mTOR complex in i-LTP and suggest that rapamycin, by selectively blocking i-LTP, represents a promising therapeutic tool to limit cellular damage after ischemic brain insult.     

Downregulation of Glutamate Transporter EAAT4 by Conditional Knockout of Rheb1 in Cerebellar Purkinje Cells

Excitatory amino acid transporter 4 (EAAT4) is believed to be critical to the synaptic activity of cerebellar Purkinje cells by limiting extracellular glutamate concentrations and facilitating the induction of long-term depression. However, the modulation of EAAT4 expression has not been elucidated. It has been shown that Ras homolog enriched in brain (Rheb)/mammalian target of rapamycin (mTOR) signaling plays essential roles in the regulation of protein translation, cell size, and cell growth. In addition, we previously found that a cascade including mTOR suppression and Akt activation induces increased expression of EAAT2 in astrocytes. In the present work, we explored whether Rheb/mTOR signaling is involved in the regulation of EAAT4 expression using conditional Rheb1 knockout mice. Our results demonstrated that Rheb1 deficiency resulted in the downregulation of EAAT4 expression, as well as decreased activity of mTOR and increased activity of Akt. The downregulation of EAAT4 was also confirmed by reduced EAAT4 currents and slowed kinetics of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor–mediated currents. On the other hand, conditional knockout of Rheb1 did not alter the morphology of Purkinje cell layer and the number of Purkinje cells. Overall, our findings suggest that small GTPase Rheb1 is a modulator in the expression of EAAT4 in Purkinje cells.     

Rapamycin is a neuroprotective treatment for traumatic brain injury

The mammalian target of rapamycin, commonly known as mTOR, is a serine/threonine kinase that regulates translation and cell division. mTOR integrates input from multiple upstream signals, including growth factors and nutrients to regulate protein synthesis. Inhibition of mTOR leads to cell cycle arrest, inhibition of cell proliferation, immunosuppression and induction of autophagy. Autophagy, a bulk degradation of sub-cellular constituents, is a process that keeps the balance between protein synthesis and protein degradation and is induced upon amino acids deprivation. Rapamycin, mTOR signaling inhibitor, mimics amino acid and, to some extent, growth factor deprivation. In the present study we examined the effect of rapamycin, on the outcome of mice after brain injury. Our results demonstrate that rapamycin injection 4 h following closed head injury significantly improved functional recovery as manifested by changes in the Neurological Severity Score, a neurobehavioral testing. To verify the activity of the injected rapamycin, we demonstrated that it inhibits p70S6K phosphorylation, reduces microglia/macrophages activation and increases the number of surviving neurons at the site of injury. We therefore suggest that rapamycin is neuroprotective following traumatic brain injury and as a drug used in the clinic for other indications, we propose that further studies on rapamycin should be conducted in order to consider it as a novel therapy for traumatic brain injury.     

Pathogenesis of tuberous sclerosis subependymal giant cell astrocytomas: biallelic inactivation of TSC1 or TSC2 leads to mTOR activation

In the central nervous system, tuberous sclerosis complex (TSC) is characterized by a range of lesions including cortical tubers, white matter heterotopias, subependymal nodules, and subependymal giant cell astrocytomas (SEGAs). Recent studies have implicated an important role for the TSC genes TSC1 and TSC2, in a signaling pathway involving the mammalian target of rapamycin (mTOR) kinase. We performed immunohistochemical and genetic analyses on SEGAs from 7 TSC patients, 4 with mutations in TSC1, and 3 with mutations in TSC2. SEGA cells show high levels of phospho-S6K, phospho-S6, and phospho-Stat3, all proteins downstream of and indicative of mTOR activation. Such expression is not seen in histologically normal control tissue. Five of 6 SEGAs also showed evidence of biallelic mutation of TSC1 or TSC2, suggesting that SEGAs develop due to complete loss of a functional tuberin-hamartin complex. We conclude that TSC SEGAs likely arise through a two-hit mechanism of biallelic inactivation of TSC1 or TSC2, leading to activation of the mTOR kinase.     

NKCC1、KCC2及mTOR相关蛋白4E-BP1在外伤性癫痫中的表达及意义

目的探讨主要表达于神经元细胞膜上的NA⁺-K⁺-2CL^-转运体（NKCC1）、K⁺-CL^-转运体（KCC2）及表达于神经元细胞质中mTOR通路信号蛋白4E结合蛋白1（4E-BP1）在外伤性癫痫（PTE）癫痫灶中的表达及临床意义。 方法收集2010年1月至2015年12月福建医科大学附属第一医院神经外科外伤性癫痫患者术后脑组织标本14例作为实验组;选取脑外伤患者行减压或清创手术获取的和各种病变患者手术入路不可避免要切除的正常脑组织8例作为对照组。用Western blot和实时定量荧光PCR（RT-PCR）检测14例PTE癫痫脑组织、8例对照组"正常脑组织"NKCC1、KCC2、4E-BP1的表达。 结果Western blot显示PTE病灶脑组织中4E-BP1、NKCC1相对灰度值（0.61±0.12、0.92±0.19）高于正常脑组织（0.27±0.05、0.67±0.66）,差异有统计学意义（P<0.05）。PTE病灶脑组织中KCC2相对灰度值（0.58±0.99）低于正常脑组织（0.72±0.06）,差异有统计学意义（P<0.05）。RT-PCR结果显示,PTE病灶脑组织中4E-BP1、NKCC1相对灰度值（30.84±1.32、27.81±1.92）高于正常脑组织（26.94±1.24、23.52±0.74）,差异有统计学意义（P<0.05）。PTE病灶脑组织中KCC2相对灰度值（21.55±1.01）低于正常脑组织（24.59±1.02）,差异有统计学意义（P<0.05）。PTE组中NKCC1/KCC2比值（1.29±0.11）高于对照组（0.96±0.26）,差异有统计学意义（P<0.05）。 结论NKCC1、KCC2和mTOR通路信号蛋白4E-BP1的异常改变,可能是外伤后脑组织组织学改变及反复癫痫发作的重要分子机制。     

Focal brain malformations: Seizures, signaling, sequencing

Focal malformations of cortical development are highly associated with intractable epilepsy in children and adults. Most patients with focal cortical malformations and epilepsy will require epilepsy surgery. Recent studies have provided new insights into the developmental pathogenesis of cortical malformations specifically relating to alterations in cell signaling though the mammalian target of rapamycin (mTOR) pathway. Focal cortical dysplasias, hemimegalencephaly, and tubers in tuberous sclerosis complex all exhibit evidence for hyperactive mTOR signaling, suggesting that these disorders form a spectrum of malformations or "TORopathies" characterized by disorganized cortical lamination, cytomegaly, and intractable seizures. Alterations in mTOR activity in focal brain malformations provide a potential pathogenic pathway to investigate for gene mutations and to exploit for animal models. Most importantly, however, if select focal cortical malformations result from enhanced mTOR signaling, new therapeutic antiepileptic compounds, such as rapamycin, can be designed and tested that specifically target mTOR signaling.     

Adipose-derived stem cells enhance myogenic differentiation in the mdx mouse model of muscular dystrophyvia paracrine signaling

Adipose-derived stem cells have been shown to promote peripheral nerve regeneration through the paracrine secretion of neurotrophic factors. However, it is unclear whether these cells can promote myogenic differentiation in muscular dystrophy. Adipose-derived stem cells(6 × 10~6) were injected into the gastrocnemius muscle of mdx mice at various sites. Dystrophin expression was found in the muscle fibers. Phosphorylation levels of Akt, mammalian target of rapamycin(mT OR), e IF-4E binding protein 1 and S6 kinase 1 were increased, and the Akt/mT OR pathway was activated. Simultaneously, myogenin levels were increased, whereas cleaved caspase 3 and vimentin levels were decreased. Necrosis and fibrosis were reduced in the muscle fibers. These findings suggest that adipose-derived stem cells promote the regeneration and survival of muscle cells by inhibiting apoptosis and fibrosis, thereby alleviating muscle damage in muscular dystrophy.     

Inhibition of mTOR Pathway by Rapamycin Decreases P-glycoprotein Expression and Spontaneous Seizures in Pharmacoresistant Epilepsy

The mammalian target of rapamycin (mTOR) has been demonstrated to mediate multidrug resistance in various tumors by inducing P-glycoprotein (P-gp) overexpression. Here, we investigated the correlation between the mTOR pathway and P-gp expression in pharmacoresistant epilepsy. Temporal cortex specimens were obtained from patients with refractory mesial temporal lobe epilepsy (mTLE) and age-matched controls who underwent surgeries at West China Hospital of Sichuan University between June 2014 and May 2015. We established a rat model of epilepsy kindled by coriaria lactone (CL) and screened pharmacoresistant rats (non-responders) using phenytoin. Non-responders were treated for 4 weeks with vehicle only or with the mTOR pathway inhibitor rapamycin at doses of 1, 3, and 6 mg/kg. Western blotting and immunohistochemistry were used to detect the expression of phospho-S6 (P-S6) and P-gp at different time points (1 h, 8 h, 1 day, 3 days, 1 weeks, 2 weeks, and 4 weeks) after the onset of treatment. Overexpression of P-S6 and P-gp was detected in both refractory mTLE patients and non-responder rats. Rapamycin showed an inhibitory effect on P-S6 and P-gp expression 1 week after treatment in rats. In addition, the expression levels of P-S6 and P-gp in the 6 mg/kg group were significantly lower than those in the 1 mg/kg or the 3 mg/kg group at the same time points (all P < 0.05). Moreover, rapamycin decreased the duration and number of CL-induced seizures, as well as the stage of non-responders (all P < 0.05). The current study indicates that the mTOR signaling pathway plays a critical role in P-gp expression in drug-resistant epilepsy. Inhibition of the mTOR pathway by rapamycin may be a potential therapeutic approach for pharmacoresistant epilepsy.     

Platelet-derived growth factor-BB restores human immunodeficiency virus Tat-cocaine-mediated impairment of neurogenesis: role of TRPC1 channels

Platelet-derived growth factor-BB (PDGF-BB) has been reported to provide tropic support for neurons in the CNS. However, whether PDGF-BB regulates neurogenesis, especially in the context of HIV-associated neurological disorder and drug abuse, remains essentially unknown. In this study, we demonstrate that pretreatment of rat hippocampal neuronal progenitor cells (NPCs) with PDGF-BB restored proliferation that had been impaired by HIV Tat-cocaine via the cognate receptors. We identify the essential role of transient receptor potential canonical (TRPC) channels in PDGF-BB-mediated proliferation. Parallel but distinct ERK/CREB, phosphatidylinositol 3-kinase/Akt signaling pathways with downstream activation of mammalian target of rapamycin (mTOR)/eukaryotic translation initiation factor 4E-binding protein (4E-BP)-p70S6K and nuclear factor-κB were critical for proliferation. Blocking TRPC1 channel suppressed PDGF-mediated proliferation as well as PDGF-BB-induced ERK/CREB and mTOR/4E-BP-p70S6K activation, thereby underscoring its role in this process. In vivo relevance of these findings was further corroborated in Tat transgenic mice wherein hippocampal injection of recombinant AAV2-PDGF-B restored impaired NPC proliferation that was induced by Tat-cocaine. Together, these data underpin the role of TRPC1 channel as a novel target that regulates cell proliferation mediated by PDGF-BB with implications for therapeutic intervention for reversal of impaired neurogenesis inflicted by Tat and cocaine.     

Rapamycin has paradoxical effects on S6 phosphorylation in rats with and without seizures

Purpose: Accumulating data have demonstrated that seizures induced by kainate (KA) or pilocarpine activate the mammalian target of rapamycin (mTOR) pathway and that mTOR inhibitor rapamycin can inhibit mTOR activation, which subsequently has potential antiepileptic effects. However, a preliminary study showed a paradoxical exacerbation of increased mTOR pathway activity reflected by S6 phosphorylation when rapamycin was administrated within a short period before KA injection. In the present study, we examined this paradoxical effect of rapamycin in more detail, both in normal rats and KA‐injected animals. Methods: Normal rats or KA‐treated rats pretreated with rapamycin at different time intervals were sacrificed at various time points (1, 3, 6, 10, 15, and 24 h) after rapamycin administration or seizure onset for western blotting analysis. Phosphorylation of mTOR signaling target of Akt, mTOR, Rictor, Raptor, S6K, and S6 were analyzed. Seizure activity was monitored behaviorally and graded according to a modified Racine scale (n = 6 for each time point). Neuronal cell death was detected by Fluoro‐Jade B staining. Key Findings: In normal rats, we found that rapamycin showed the expected dose‐dependent inhibition of S6 phosphorylation 3–24 h after injection, whereas a paradoxical elevation of S6 phosphorylation was observed 1 h after rapamycin. Similarly, pretreatment with rapamycin over 10 h before KA inhibited the KA seizure–induced mTOR activation. In contrast, rapamycin administered 1–6 h before KA caused a paradoxical increase in the KA seizure–induced mTOR activation. Rats pretreated with rapamycin 1 h prior to KA exhibited an increase in severity and duration of seizures and more neuronal cell death as compared to vehicle‐treated groups. In contrast, rapamycin pretreated 10 h prior to KA had no effect on the seizures and decreased neuronal cell death. The paradoxical effect of rapamycin on S6 phosphorylation was correlated with upstream mTOR signaling and was reversed by pretreatment of perifosine, an Akt inhibitor. Significance: These data indicate the complexity of S6 regulation and its effect on epilepsy. Paradoxical effects of rapamycin need to be considered in clinical applications, such as for potential treatment for epilepsy and other neurologic disorders.     

Acetaldehyde promotes rapamycin-dependent activation of p70(S6K) and glucose uptake despite inhibition of Akt and mTOR in dopaminergic SH-SY5Y human neuroblastoma cells

Alcohol intake is one of the important lifestyle factors for the risk of insulin resistance and type 2 diabetes. Acetaldehyde, the major ethanol metabolite which is far more reactive than ethanol, has been postulated to participate in alcohol-induced tissue injury although its direct impact on insulin signaling is unclear. This study was designed to examine the effect of acetaldehyde on glucose uptake and insulin signaling in human dopaminergic SH-SY5Y cells. Akt, mammalian target of rapamycin (mTOR), ribosomal-S6 kinase (p70(S6K)), the eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1) and insulin receptor substrate (IRS)-2 were evaluated by Western blot analysis. Glucose uptake and apoptosis were measured using [(3)H]-2-deoxyglucose uptake and caspase-3 assay, respectively. Short-term exposure (12 h) of acetaldehyde (150 muM) facilitated glucose uptake in a rapamycin-dependent manner without affecting apoptosis, IRS-2 expression and insulin-stimulated glucose uptake in SH-SY5Y cells. Acetaldehyde suppressed basal and insulin-stimulated Akt phosphorylation without affecting total Akt expression. Acetaldehyde inhibited mTOR phosphorylation without affecting total mTOR and insulin-elicited response on mTOR phosphorylation. Rapamycin, which inhibits mTOR leading to inactivation of p70(S6K), did not affect acetaldehyde-induced inhibition on phosphorylation of Akt and mTOR. Interestingly, acetaldehyde enhanced p70(S6K) activation and depressed 4E-BP1 phosphorylation, the effect of which was blunted and exaggerated, respectively, by rapamycin. Collectively, these data suggested that acetaldehyde did not adversely affect glucose uptake despite inhibition of insulin signaling cascade at the levels of Akt and mTOR, possibly due to presence of certain mechanism(s) responsible for enhanced p70(S6K) phosphorylation.