Elongation factor 1A1 regulates metabolic substrate preference in mammalian cells.

Eukaryotic elongation factor 1A1 (EEF1A1) is canonically involved in protein synthesis but also has noncanonical functions in diverse cellular processes. Previously, we identified EEF1A1 as a mediator of lipotoxicity and demonstrated that chemical inhibition of EEF1A1 activity reduced mouse liver lipid accumulation. These findings suggested a link between EEF1A1 and metabolism. Therefore, we investigated its role in regulating metabolic substrate preference. EEF1A1-deficient Chinese hamster ovary (2E2) cells displayed reduced media lactate accumulation. These effects were also observed with EEF1A1 knockdown in human hepatocyte-like HepG2 cells and in WT Chinese hamster ovary and HepG2 cells treated with selective EEF1A inhibitors, didemnin B, or plitidepsin. Extracellular flux analyses revealed decreased glycolytic ATP production and increased mitochondrial-to-glycolytic ATP production ratio in 2E2 cells, suggesting a more oxidative metabolic phenotype. Correspondingly, fatty acid oxidation was increased in 2E2 cells. Both 2E2 cells and HepG2 cells treated with didemnin B exhibited increased neutral lipid content, which may be required to support elevated oxidative metabolism. RNA-seq revealed a >90-fold downregulation of a rate-limiting glycolytic enzyme, hexokinase 2, which we confirmed through immunoblotting and enzyme activity assays. Pathway enrichment analysis identified downregulations in TNFA signaling via NFKB and MYC targets. Correspondingly, nuclear abundances of RELB and MYC were reduced in 2E2 cells. Thus, EEF1A1 deficiency may perturb glycolysis by limiting NFKB- and MYC-mediated gene expression, leading to decreased hexokinase expression and activity. This is the first evidence of a role for a translation elongation factor, EEF1A1, in regulating metabolic substrate utilization in mammalian cells.

Abnormal Social Interactions in a Drosophila Mutant of an Autism Candidate Gene: Neuroligin 3.

Social interactions are typically impaired in neuropsychiatric disorders such as autism, for which the genetic underpinnings are very complex. Social interactions can be modeled by analysis of behaviors, including social spacing, sociability, and aggression, in simpler organisms such as Drosophila melanogaster. Here, we examined the effects of mutants of the autism-related gene neuroligin 3 (nlg3) on fly social and non-social behaviors. Startled-induced negative geotaxis is affected by a loss of function nlg3 mutation. Social space and aggression are also altered in a sex- and social-experience-specific manner in nlg3 mutant flies. In light of the conserved roles that neuroligins play in social behavior, our results offer insight into the regulation of social behavior in other organisms, including humans.

The association between oxidative stress-induced galectins and differentiation of human promyelocytic HL-60 cells.

Galectins are multifunctional ß-galactoside-binding proteins that are involved in the regulation of cellular stress responses and differentiation. The relationship between these processes is unclear and we report here that galectins display oxidative-stress specific expression patterns in neutrophil-like differentiated HL-60 cells. Three galectins (-1, -3, and -10) are upregulated in response to either menadione or DMSO exposure whereas galectins -9 and -12 exhibited a stimulus-dependent downregulation. Changes in galectin expression are oxidant dependent based on the observations that 1) oxidative stress biomarkers HMOX1 (heme oxygenase-1) and NCF1 (neutrophil cytosolic factor 1, which is also a biomarker of neutrophil differentiation) are elevated in both cases, and 2) the antioxidant N-acetyl-L-cysteine restores basal expression of galectin-3 following oxidant exposure. In addition, our results suggest that the regulation of oxidative stress-sensitive galectins involves DNA hypomethylation mechanisms. Expression of galectin-3 and galectin-12 exhibits an opposite relationship to the expression of HMOX1/NCF1, suggesting a stimulatory and inhibitory role of these galectins in neutrophil-like differentiation of HL-60 cells. We also show that the inhibition of galectins reduces the growth rate of HL-60 cells, and facilitates their neutrophil-like differentiation. Collectively, our findings indicate that the process of cellular differentiation implicates, in part, oxidative stress-sensitive galectins, which further highlights a biological significance of galectin network remodeling in cells.

Aerobic Glycolysis in the Frontal Cortex Correlates with Memory Performance in Wild-Type Mice But Not the APP/PS1 Mouse Model of Cerebral Amyloidosis.

Aerobic glycolysis and lactate production in the brain plays a key role in memory, yet the role of this metabolism in the cognitive decline associated with Alzheimer's disease (AD) remains poorly understood. Here we examined the relationship between cerebral lactate levels and memory performance in an APP/PS1 mouse model of AD, which progressively accumulates amyloid-ß. In vivo (1)H-magnetic resonance spectroscopy revealed an age-dependent decline in lactate levels within the frontal cortex of control mice, whereas lactate levels remained unaltered in APP/PS1 mice from 3 to 12 months of age. Analysis of hippocampal interstitial fluid by in vivo microdialysis revealed a significant elevation in lactate levels in APP/PS1 mice relative to control mice at 12 months of age. An age-dependent decline in the levels of key aerobic glycolysis enzymes and a concomitant increase in lactate transporter expression was detected in control mice. Increased expression of lactate-producing enzymes correlated with improved memory in control mice. Interestingly, in APP/PS1 mice the opposite effect was detected. In these mice, increased expression of lactate producing enzymes correlated with poorer memory performance. Immunofluorescent staining revealed localization of the aerobic glycolysis enzymes pyruvate dehydrogenase kinase and lactate dehydrogenase A within cortical and hippocampal neurons in control mice, as well as within astrocytes surrounding amyloid plaques in APP/PS1 mice. These observations collectively indicate that production of lactate, via aerobic glycolysis, is beneficial for memory function during normal aging. However, elevated lactate levels in APP/PS1 mice indicate perturbed lactate processing, a factor that may contribute to cognitive decline in AD. SIGNIFICANCE STATEMENT: Lactate has recently emerged as a key metabolite necessary for memory consolidation. Lactate is the end product of aerobic glycolysis, a unique form of metabolism that occurs within certain regions of the brain. Here we detected an age-dependent decline in the expression of aerobic glycolysis enzymes and a concomitant decrease in lactate levels within the frontal cortex of wild-type mice. Improved memory performance in wild-type mice correlated with elevated expression of aerobic glycolysis enzymes. Surprisingly, lactate levels remained elevated with age and increased aerobic glycolysis enzyme expression correlated with poorer memory performance in APP/PS1 mice. These findings suggest that while lactate production is beneficial for memory in the healthy aging brain, it might be detrimental in an Alzheimer's disease context.

Age-dependent metabolic dysregulation in cancer and Alzheimer's disease.

Age is the main risk factor for cancer and neurodegeneration; two radically divergent diseases. Yet selective pressure to meet cellular metabolic needs may provide a common mechanism linking these two disorders. The exclusive use of glycolysis, despite the presence of oxygen, is commonly referred to as aerobic glycolysis and is the primary metabolic pathway of cancer cells. Recent evidence suggests that aerobic glycolysis is also a key regulator of synaptic plasticity in the brain that may positively influence cognition. Elevated aerobic glycolysis is a contributing factor to the development of cancer as increased glycolytic flux plays an important role in the biosynthesis of macromolecules and promotes proliferation. In contrast, decreased aerobic glycolysis in the brain occurs with age and could lead to a loss of cell survival mechanisms that counter pathogenic processes underlying neurodegeneration. In this review we discuss the recent findings from epidemiological studies demonstrating an inverse comorbidity of cancer and Alzheimer's disease. We summarize evidence linking the two diseases through changes in metabolism over the course of normal aging. We discuss the key steps and regulatory mechanisms of aerobic glycolysis and mitochondrial oxidative phosphorylation which could be exploited for the development of novel therapies. In addition, we outline the regulation of aerobic glycolysis at the transcriptional level by HIF-1α and Pin1 and their roles in cancer and neurodegeneration. Finally, we provide a possible explanation for metabolic dysregulation that occurs with age, and how it may be a contributing factor to age-related diseases. Determining how metabolism becomes dysregulated over time could lead to the development of effective interventions for ensuring metabolic homeostasis and healthy aging.

Overexpression of pyruvate dehydrogenase kinase 1 and lactate dehydrogenase A in nerve cells confers resistance to amyloid ß and other toxins by decreasing mitochondrial respiration and reactive oxygen species production.

We previously demonstrated that nerve cell lines selected for resistance to amyloid ß (Aß) peptide exhibit elevated aerobic glycolysis in part due to increased expression of pyruvate dehydrogenase kinase 1 (PDK1) and lactate dehydrogenase A (LDHA). Here, we show that overexpression of either PDK1 or LDHA in a rat CNS cell line (B12) confers resistance to Aß and other neurotoxins. Treatment of Aß-sensitive cells with various toxins resulted in mitochondrial hyperpolarization, immediately followed by rapid depolarization and cell death, events accompanied by increased production of cellular reactive oxygen species (ROS). In contrast, cells expressing either PDK1 or LDHA maintained a lower mitochondrial membrane potential and decreased ROS production with or without exposure to toxins. Additionally, PDK1- and LDHA-overexpressing cells exhibited decreased oxygen consumption but maintained levels of ATP under both normal culture conditions and following Aß treatment. Interestingly, immunoblot analysis of wild type mouse primary cortical neurons treated with Aß or cortical tissue extracts from 12-month-old APPswe/PS1dE9 transgenic mice showed decreased expression of LDHA and PDK1 when compared with controls. Additionally, post-mortem brain extracts from patients with Alzheimer disease exhibited a decrease in PDK1 expression compared with nondemented patients. Collectively, these findings indicate that key Warburg effect enzymes play a central role in mediating neuronal resistance to Αß or other neurotoxins by decreasing mitochondrial activity and subsequent ROS production. Maintenance of PDK1 or LDHA expression in certain regions of the brain may explain why some individuals tolerate high levels of Aß deposition without developing Alzheimer disease.

Dithiol-based compounds maintain expression of antioxidant protein peroxiredoxin 1 that counteracts toxicity of mutant huntingtin.

Mitochondrial dysfunction and elevated reactive oxygen species are strongly implicated in both aging and various neurodegenerative disorders, including Huntington disease (HD). Because reactive oxygen species can promote the selective oxidation of protein cysteine sulfhydryl groups to disulfide bonds we examined the spectrum of disulfide-bonded proteins that were specifically altered in a HD context. Protein extracts from PC12 cells overexpressing the amino-terminal fragment of the Huntingtin (Htt) protein with either a nonpathogenic or pathogenic polyglutamine repeat (Htt-103Q) were resolved by redox two-dimensional PAGE followed by mass spectrometry analysis. Several antioxidant proteins were identified that exhibited changes in disulfide bonding unique to Htt-103Q expressing cells. In particular, the antioxidant protein peroxiredoxin 1 (Prx1) exhibited both decreased expression and hyperoxidation in response to mutant Htt expressed in either PC12 cells or immortalized striatal cells exposed to 3-nitropropionic acid. Ectopic expression of Prx1 in PC12 cells attenuated mutant Htt-induced toxicity. In contrast, short hairpin RNA-mediated knockdown of Prx1 potentiated mHtt toxicity. Furthermore, treatment with the dithiol-based compounds dimercaptopropanol and dimercaptosuccinic acid suppressed toxicity in both HD cell models, whereas monothiol compounds were relatively ineffective. Dimercaptopropanol treatment also prevented mutant Htt-induced loss of Prx1 expression in both cell models. Our studies reveal for the first time that pathogenic Htt can affect the expression and redox state of antioxidant proteins; an event countered by specific dithiol-based compounds. These findings should provide a catalyst to explore the use of dithiol-based drugs for the treatment of neurodegenerative diseases.

Aging and memory are altered by genetically manipulating lactate dehydrogenase in the neurons or glia of flies.

The astrocyte-neuron lactate shuttle hypothesis posits that glial-generated lactate is transported to neurons to fuel metabolic processes required for long-term memory. Although studies in vertebrates have revealed that lactate shuttling is important for cognitive function, it is uncertain if this form of metabolic coupling is conserved in invertebrates or is influenced by age. Lactate dehydrogenase (Ldh) is a rate limiting enzyme that interconverts lactate and pyruvate. Here we genetically manipulated expression of Drosophila melanogaster lactate dehydrogenase (dLdh) in neurons or glia to assess the impact of altered lactate metabolism on invertebrate aging and long-term courtship memory at different ages. We also assessed survival, negative geotaxis, brain neutral lipids (the core component of lipid droplets) and brain metabolites. Both upregulation and downregulation of dLdh in neurons resulted in decreased survival and memory impairment with age. Glial downregulation of dLdh expression caused age-related memory impairment without altering survival, while upregulated glial dLdh expression lowered survival without disrupting memory. Both neuronal and glial dLdh upregulation increased neutral lipid accumulation. We provide evidence that altered lactate metabolism with age affects the tricarboxylic acid (TCA) cycle, 2-hydroxyglutarate (2HG), and neutral lipid accumulation. Collectively, our findings indicate that the direct alteration of lactate metabolism in either glia or neurons affects memory and survival but only in an age-dependent manner.

Deletion of p66Shc Dysregulates ERK and STAT3 Activity in Mouse Embryonic Stem Cells, Enhancing Their Naive-Like Self-Renewal in the Presence of Leukemia Inhibitory Factor.

The ShcA adapter protein is necessary for early embryonic development. The role of ShcA in development is primarily attributed to its 52 and 46 kDa isoforms that transduce receptor tyrosine kinase signaling through the extracellular signal regulated kinase (ERK). During embryogenesis, ERK acts as the primary signaling effector, driving fate acquisition and germ layer specification. P66Shc, the largest of the ShcA isoforms, has been observed to antagonize ERK in several contexts; however, its role during embryonic development remains poorly understood. We hypothesized that p66Shc could act as a negative regulator of ERK activity during embryonic development, antagonizing early lineage commitment. To explore the role of p66Shc in stem cell self-renewal and differentiation, we created a p66Shc knockout murine embryonic stem cell (mESC) line. Deletion of p66Shc enhanced basal ERK activity, but surprisingly, instead of inducing mESC differentiation, loss of p66Shc enhanced the expression of core and naive pluripotency markers. Using pharmacologic inhibitors to interrogate potential signaling mechanisms, we discovered that p66Shc deletion permits the self-renewal of naive mESCs in the absence of conventional growth factors, by increasing their responsiveness to leukemia inhibitory factor (LIF). We discovered that loss of p66Shc enhanced not only increased ERK phosphorylation but also increased phosphorylation of Signal transducer and activator of transcription in mESCs, which may be acting to stabilize their naive-like identity, desensitizing them to ERK-mediated differentiation cues. These findings identify p66Shc as a regulator of both LIF-mediated ESC pluripotency and of signaling cascades that initiate postimplantation embryonic development and ESC commitment.

Using the urea cycle to shift astrocytes from harmful to helpful in Alzheimer's disease.

Astrocytes are brain cells that react to Alzheimer's disease pathology in ways that can have either beneficial or detrimental effects. In this issue of Cell Metabolism, Ju et al. outline a novel strategy for coercing astrocytes to a neuroprotective state by maintaining liver-like detoxification in the brain without producing damaging byproducts.

Lactate preconditioning promotes a HIF-1α-mediated metabolic shift from OXPHOS to glycolysis in normal human diploid fibroblasts.

Recent evidence has emerged that cancer cells can use various metabolites as fuel sources. Restricting cultured cancer cells to sole metabolite fuel sources can promote metabolic changes leading to enhanced glycolysis or mitochondrial OXPHOS. However, the effect of metabolite-restriction on non-transformed cells remains largely unexplored. Here we examined the effect of restricting media fuel sources, including glucose, pyruvate or lactate, on the metabolic state of cultured human dermal fibroblasts. Fibroblasts cultured in lactate-only medium exhibited reduced PDH phosphorylation, indicative of OXPHOS, and a concurrent elevation of ROS. Lactate exposure primed fibroblasts to switch to glycolysis by increasing transcript abundance of genes encoding glycolytic enzymes and, upon exposure to glucose, increasing glycolytic enzyme levels. Furthermore, lactate treatment stabilized HIF-1α, a master regulator of glycolysis, in a manner attenuated by antioxidant exposure. Our findings indicate that lactate preconditioning primes fibroblasts to switch from OXPHOS to glycolysis metabolism, in part, through ROS-mediated HIF-1α stabilization. Interestingly, we found that lactate preconditioning results in increased transcript abundance of MYC and SNAI1, key facilitators of early somatic cell reprogramming. Defined metabolite treatment may represent a novel approach to increasing somatic cell reprogramming efficiency by amplifying a critical metabolic switch that occurs during iPSC generation.

Importance of collecting data on socioeconomic determinants from the early stage of the COVID-19 outbreak onwards.

Disadvantaged socioeconomic position (SEP) is widely associated with disease and mortality, and there is no reason to think this will not be the case for the newly emerged coronavirus disease 2019 (COVID-19) that has reached a pandemic level. Individuals with a more disadvantaged SEP are more likely to be affected by most of the known risk factors of COVID-19. SEP has been previously established as a potential determinant of infectious diseases in general. We hypothesise that SEP plays an important role in the COVID-19 pandemic either directly or indirectly via occupation, living conditions, health-related behaviours, presence of comorbidities and immune functioning. However, the influence of socioeconomic factors on COVID-19 transmission, severity and outcomes is not yet known and is subject to scrutiny and investigation. Here we briefly review the extent to which SEP has been considered as one of the potential risk factors of COVID-19. From 29 eligible studies that reported the characteristics of patients with COVID-19 and their potential risk factors, only one study reported the occupational position of patients with mild or severe disease. This brief overview of the literature highlights that important socioeconomic characteristics are being overlooked when data are collected. As COVID-19 spreads worldwide, it is crucial to collect and report data on socioeconomic determinants as well as race/ethnicity to identify high-risk populations. A systematic recording of socioeconomic characteristics of patients with COVID-19 will be beneficial to identify most vulnerable groups, to identify how SEP relates to COVID-19 and to develop equitable public health prevention measures, guidelines and interventions.

Lactate dehydrogenase expression modulates longevity and neurodegeneration in Drosophila melanogaster.

Lactate dehydrogenase (LDH) catalyzes the conversion of glycolysis-derived pyruvate to lactate. Lactate has been shown to play key roles in brain energetics and memory formation. However, lactate levels are elevated in aging and Alzheimer's disease patients, and it is not clear whether lactate plays protective or detrimental roles in these contexts. Here we show that Ldh transcript levels are elevated and cycle with diurnal rhythm in the heads of aged flies and this is associated with increased LDH protein, enzyme activity, and lactate concentrations. To understand the biological significance of increased Ldh gene expression, we genetically manipulated Ldh levels in adult neurons or glia. Overexpression of Ldh in both cell types caused a significant reduction in lifespan whereas Ldh down-regulation resulted in lifespan extension. Moreover, pan-neuronal overexpression of Ldh disrupted circadian locomotor activity rhythms and significantly increased brain neurodegeneration. In contrast, reduction of Ldh in neurons delayed age-dependent neurodegeneration. Thus, our unbiased genetic approach identified Ldh and lactate as potential modulators of aging and longevity in flies.

Simple Protocol for Distinguishing Drug-induced Effects on Spatial Memory Acquisition, Consolidation and Retrieval in Mice Using the Morris Water Maze.

The Morris water maze (MWM) is one of the most commonly used tests for assessing spatial learning and memory in mice. While the MWM is highly amenable to testing the effects of memory modifying drugs, most studies do not consider the timing or duration of drug exposure when conducting the MWM assay; factors that can strongly influence the effect of the drug on different stages of memory and interfere with data interpretation. Herein we describe a MWM protocol which offers the advantage of distinguishing the impact of a fast acting intraperitoneally (IP) injected drug on the different stages of spatial memory: acquisition, consolidation, and retrieval. Mice initially undergo habituation to both the MWM apparatus and IP injection procedure over the course of three days. For assessing the effect of a drug on memory acquisition, mice are injected with the drug prior to training sessions over four consecutive days, where mice learn to find an escape platform in a circular water tank using distal spatial cues. To determine the effect of the drug on memory consolidation, mice are injected with the drug immediately after each training session. For testing the effect of a drug on memory retrieval, mice receive mock IP injections on each training day and the drug is IP injected only once, prior to a probe trial, where mice attempt to locate the platform following its removal from the tank. This protocol provides a simple strategy for distinguishing the effect(s) of a CNS acting drug on the different stages of memory.

Aerobic Glycolysis Is Required for Spatial Memory Acquisition But Not Memory Retrieval in Mice.

The consolidation of newly formed memories and their retrieval are energetically demanding processes. Aerobic glycolysis (AG), also known as the Warburg effect, consists of the production of lactate from glucose in the presence of oxygen. The astrocyte neuron lactate shuttle hypothesis posits that astrocytes process glucose by AG to generate lactate, which is used as a fuel source within neurons to maintain synaptic activity. Studies in mice have demonstrated that lactate transport between astrocytes and neurons is required for long-term memory formation, yet the role of lactate production in memory acquisition and retrieval has not previously been explored. Here, we examined the effect of dichloroacetate (DCA), a chemical inhibitor of lactate production, on spatial learning and memory in mice using the Morris water maze (MWM). In vivo hyperpolarized 13C-pyruvate magnetic resonance spectroscopy revealed decreased conversion of pyruvate to lactate in the mouse brain following DCA administration, concomitant with a reduction in the phosphorylation of pyruvate dehydrogenase. DCA exposure before each training session in the MWM impaired learning, which subsequently resulted in impaired memory during the probe trial. In contrast, mice that underwent training without DCA exposure, but received a single DCA injection before the probe trial exhibited normal memory. Our findings indicate that AG plays a key role during memory acquisition but is less important for the retrieval of established memories. Thus, the activation of AG may be important for learning-dependent synaptic plasticity rather than the activation of signaling cascades required for memory retrieval.

Genetic modifiers of the Drosophila blue cheese gene link defects in lysosomal transport with decreased life span and altered ubiquitinated-protein profiles.

Defects in lysosomal trafficking pathways lead to decreased cell viability and are associated with progressive disorders in humans. Previously we have found that loss-of-function (LOF) mutations in the Drosophila gene blue cheese (bchs) lead to reduced adult life span, increased neuronal death, and widespread CNS degeneration that is associated with the formation of ubiquitinated-protein aggregates. To identify potential genes that participate in the bchs functional pathway, we conducted a genetic modifier screen based on alterations of an eye phenotype that arises from high-level overexpression of Bchs. We found that mutations in select autophagic and endocytic trafficking genes, defects in cytoskeletal and motor proteins, as well as mutations in the SUMO and ubiquitin signaling pathways behave as modifiers of the Bchs gain-of-function (GOF) eye phenotype. Individual mutant alleles that produced viable adults were further examined for bchs-like phenotypes. Mutations in several lysosomal trafficking genes resulted in significantly decreased adult life spans and several mutants showed changes in ubiquitinated protein profiles as young adults. This work represents a novel approach to examine the role that lysosomal transport and function have on adult viability. The genes characterized in this study have direct human homologs, suggesting that similar defects in lysosomal transport may play a role in human health and age-related processes.

Analysis of global and specific changes in the disulfide proteome using redox two-dimensional polyacrylamide gel electrophoresis.

Protein cysteine sulfhydryl groups are susceptible to a number of redox-dependent modifications, including an interchange between the reduced sulfhydryl and an oxidized disulfide state. A growing body of evidence suggests that reversible disulfide bond formation alters the structure and function of proteins. In this chapter, a method is described for isolating disulfide bonded proteins from different subcellular compartments by using a differential detergent fractionation technique followed by sequential nonreducing/reducing two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (i.e., Redox 2D-PAGE). This method can be adapted to examine individual redox-active proteins by immunoprecipitating an epitope-tagged redox protein expressed in cultured cells and using Redox 2D-PAGE and mass spectrometry to identify proteins that form mixed disulfides with the tagged protein. With the use of these techniques, it is shown that disulfide bond formation occurs within families of cytoplasmic proteins and may provide a common mechanism used to control multiple physiological processes.

p66Shc activation promotes increased oxidative phosphorylation and renders CNS cells more vulnerable to amyloid beta toxicity.

A key pathological feature of Alzheimer's disease (AD) is the accumulation of the neurotoxic amyloid beta (Aß) peptide within the brains of affected individuals. Previous studies have shown that neuronal cells selected for resistance to Aß toxicity display a metabolic shift from mitochondrial-dependent oxidative phosphorylation (OXPHOS) to aerobic glycolysis to meet their energy needs. The Src homology/collagen (Shc) adaptor protein p66Shc is a key regulator of mitochondrial function, ROS production and aging. Moreover, increased expression and activation of p66Shc promotes a shift in the cellular metabolic state from aerobic glycolysis to OXPHOS in cancer cells. Here we evaluated the hypothesis that activation of p66Shc in CNS cells promotes both increased OXPHOS and enhanced sensitivity to Aß toxicity. The effect of altered p66Shc expression on metabolic activity was assessed in rodent HT22 and B12 cell lines of neuronal and glial origin respectively. Overexpression of p66Shc repressed glycolytic enzyme expression and increased both mitochondrial electron transport chain activity and ROS levels in HT22 cells. The opposite effect was observed when endogenous p66Shc expression was knocked down in B12 cells. Moreover, p66Shc activation in both cell lines increased their sensitivity to Aß toxicity. Our findings indicate that expression and activation of p66Shc renders CNS cells more sensitive to Aß toxicity by promoting mitochondrial OXPHOS and ROS production while repressing aerobic glycolysis. Thus, p66Shc may represent a potential therapeutically relevant target for the treatment of AD.

Effects of Global O-GlcNAcylation on Galectin Gene-expression Profiles in Human Cancer Cell Lines.

BACKGROUND/AIM: The effects of O-linked ß-N-acetyl-D-glucosamine (O-GlcNAc) transferase (OGT) and O-GlcNAcase (OGA) inhibitors on galectin gene expression profiles were examined in MCF7, HT-29, and HL-60 cancer cell lines. MATERIALS AND METHODS: Cell cultures were treated for 24 h with OGA inhibitor thiamet G or OGT inhibitor 2-acetamido-1,3,4,6-tetra-O-acetyl-2-deoxy-5-thio-α-D-glucopyranose, and global O-GlcNAc levels and expression of galectin genes were determined using an immunodot blot assay and real-time quantitative polymerase chain reaction. RESULTS: Two galectin genes, LGALS3 in MCF7 cells and LGALS12 in HL-60 cells, were up-regulated by O-GlcNAc, whereas other cell-specific galectins were unresponsive to changes in O-GlcNAc level. Of interest, basal levels of O-GlcNAc in resting HL-60 and HT-29 cells were significantly higher than those in cells differentiated into neutrophilic or enterocytic lineages, respectively. CONCLUSION: O-GlcNAc-mediated signaling pathways may be involved in regulating the expression of only a limited number of galectin genes. Additional O-GlcNAc-dependent mechanisms may work at the protein level (galectin secretion and intracellular localization) and warrant further investigation.

Amyloid-beta induces disulfide bonding and aggregation of GAPDH in Alzheimer's disease.

GAPDH is a redox-sensitive glycolytic enzyme that also promotes apoptosis when translocated to the nucleus and associates with aggregate-prone proteins involved in neurodegenerative disorders. Recent evidence indicates that polymorphic variation within GAPDH genes is associated with an elevated risk of developing Alzheimer's disease (AD). We previously demonstrated that GAPDH readily undergoes disulfide bonding following oxidant exposure, although the consequence of disulfide bonding on GAPDH activity or function is unknown. Here we show that increased GAPDH disulfide bonding is observed in detergent-insoluble extracts from AD patient and transgenic AD mouse brain tissue compared with age-matched controls. Exposure of primary rat cortical neurons to the pro-oxidant amyloid beta peptide promotes nuclear accumulation of a disulfide-linked form of GAPDH, which becomes detergent-insoluble. Disulfide bonding leads to a reduction in GAPDH enzymatic activity and correlates with the appearance of punctate aggregate-like GAPDH staining within the cytoplasm of both oxidant-treated HT22 cells and amyloid beta-treated primary cortical neurons. Our findings suggest that disulfide bonding of GAPDH and subsequent protein aggregate formation may have relevance to the pathophysiology of AD.