Niche-Selective Inhibition of Pathogenic Th17 Cells by Targeting Metabolic Redundancy.

Targeting glycolysis has been considered therapeutically intractable owing to its essential housekeeping role. However, the context-dependent requirement for individual glycolytic steps has not been fully explored. We show that CRISPR-mediated targeting of glycolysis in T cells in mice results in global loss of Th17 cells, whereas deficiency of the glycolytic enzyme glucose phosphate isomerase (Gpi1) selectively eliminates inflammatory encephalitogenic and colitogenic Th17 cells, without substantially affecting homeostatic microbiota-specific Th17 cells. In homeostatic Th17 cells, partial blockade of glycolysis upon Gpi1 inactivation was compensated by pentose phosphate pathway flux and increased mitochondrial respiration. In contrast, inflammatory Th17 cells experience a hypoxic microenvironment known to limit mitochondrial respiration, which is incompatible with loss of Gpi1. Our study suggests that inhibiting glycolysis by targeting Gpi1 could be an effective therapeutic strategy with minimum toxicity for Th17-mediated autoimmune diseases, and, more generally, that metabolic redundancies can be exploited for selective targeting of disease processes.

BACH1 Stabilization by Antioxidants Stimulates Lung Cancer Metastasis.

For tumors to progress efficiently, cancer cells must overcome barriers of oxidative stress. Although dietary antioxidant supplementation or activation of endogenous antioxidants by NRF2 reduces oxidative stress and promotes early lung tumor progression, little is known about its effect on lung cancer metastasis. Here, we show that long-term supplementation with the antioxidants N-acetylcysteine and vitamin E promotes KRAS-driven lung cancer metastasis. The antioxidants stimulate metastasis by reducing levels of free heme and stabilizing the transcription factor BACH1. BACH1 activates transcription of Hexokinase 2 and Gapdh and increases glucose uptake, glycolysis rates, and lactate secretion, thereby stimulating glycolysis-dependent metastasis of mouse and human lung cancer cells. Targeting BACH1 normalized glycolysis and prevented antioxidant-induced metastasis, while increasing endogenous BACH1 expression stimulated glycolysis and promoted metastasis, also in the absence of antioxidants. We conclude that BACH1 stimulates glycolysis-dependent lung cancer metastasis and that BACH1 is activated under conditions of reduced oxidative stress.

Mitochondrial translation deficiency impairs NAD+ -mediated lysosomal acidification.

Mitochondrial translation dysfunction is associated with neurodegenerative and cardiovascular diseases. Cells eliminate defective mitochondria by the lysosomal machinery via autophagy. The relationship between mitochondrial translation and lysosomal function is unknown. In this study, mitochondrial translation-deficient hearts from p32-knockout mice were found to exhibit enlarged lysosomes containing lipofuscin, suggesting impaired lysosome and autolysosome function. These mice also displayed autophagic abnormalities, such as p62 accumulation and LC3 localization around broken mitochondria. The expression of genes encoding for nicotinamide adenine dinucleotide (NAD+ ) biosynthetic enzymes-Nmnat3 and Nampt-and NAD+ levels were decreased, suggesting that NAD+ is essential for maintaining lysosomal acidification. Conversely, nicotinamide mononucleotide (NMN) administration or Nmnat3 overexpression rescued lysosomal acidification. Nmnat3 gene expression is suppressed by HIF1α, a transcription factor that is stabilized by mitochondrial translation dysfunction, suggesting that HIF1α-Nmnat3-mediated NAD+ production is important for lysosomal function. The glycolytic enzymes GAPDH and PGK1 were found associated with lysosomal vesicles, and NAD+ was required for ATP production around lysosomal vesicles. Thus, we conclude that NAD+ content affected by mitochondrial dysfunction is essential for lysosomal maintenance.

Tyr-Asp inhibition of glyceraldehyde 3-phosphate dehydrogenase affects plant redox metabolism.

How organisms integrate metabolism with the external environment is a central question in biology. Here, we describe a novel regulatory small molecule, a proteogenic dipeptide Tyr-Asp, which improves plant tolerance to oxidative stress by directly interfering with glucose metabolism. Specifically, Tyr-Asp inhibits the activity of a key glycolytic enzyme, glyceraldehyde 3-phosphate dehydrogenase (GAPC), and redirects glucose toward pentose phosphate pathway (PPP) and NADPH production. In line with the metabolic data, Tyr-Asp supplementation improved the growth performance of both Arabidopsis and tobacco seedlings subjected to oxidative stress conditions. Moreover, inhibition of Arabidopsis phosphoenolpyruvate carboxykinase (PEPCK) activity by a group of branched-chain amino acid-containing dipeptides, but not by Tyr-Asp, points to a multisite regulation of glycolytic/gluconeogenic pathway by dipeptides. In summary, our results open the intriguing possibility that proteogenic dipeptides act as evolutionarily conserved small-molecule regulators at the nexus of stress, protein degradation, and metabolism.

GAPDH mediates drug resistance and metabolism in Plasmodium falciparum malaria parasites.

Efforts to control the global malaria health crisis are undermined by antimalarial resistance. Identifying mechanisms of resistance will uncover the underlying biology of the Plasmodium falciparum malaria parasites that allow evasion of our most promising therapeutics and may reveal new drug targets. We utilized fosmidomycin (FSM) as a chemical inhibitor of plastidial isoprenoid biosynthesis through the methylerythritol phosphate (MEP) pathway. We have thus identified an unusual metabolic regulation scheme in the malaria parasite through the essential glycolytic enzyme, glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Two parallel genetic screens converged on independent but functionally analogous resistance alleles in GAPDH. Metabolic profiling of FSM-resistant gapdh mutant parasites indicates that neither of these mutations disrupt overall glycolytic output. While FSM-resistant GAPDH variant proteins are catalytically active, they have reduced assembly into the homotetrameric state favored by wild-type GAPDH. Disrupted oligomerization of FSM-resistant GAPDH variant proteins is accompanied by altered enzymatic cooperativity and reduced susceptibility to inhibition by free heme. Together, our data identifies a new genetic biomarker of FSM-resistance and reveals the central role of GAPDH in MEP pathway control and antimalarial sensitivity.

The Immunomodulatory Enzyme IDO2 Mediates Autoimmune Arthritis through a Nonenzymatic Mechanism.

IDO2 is one of two closely related tryptophan catabolizing enzymes induced under inflammatory conditions. In contrast to the immunoregulatory role defined for IDO1 in cancer models, IDO2 has a proinflammatory function in models of autoimmunity and contact hypersensitivity. In humans, two common single-nucleotide polymorphisms have been identified that severely impair IDO2 enzymatic function, such that <25% of individuals express IDO2 with full catalytic potential. This, together with IDO2's relatively weak enzymatic activity, suggests that IDO2 may have a role outside of its function in tryptophan catabolism. To determine whether the enzymatic activity of IDO2 is required for its proinflammatory function, we used newly generated catalytically inactive IDO2 knock-in mice together with established models of contact hypersensitivity and autoimmune arthritis. Contact hypersensitivity was attenuated in catalytically inactive IDO2 knock-in mice. In contrast, induction of autoimmune arthritis was unaffected by the absence of IDO2 enzymatic activity. In pursuing this nonenzymatic IDO2 function, we identified GAPDH, Runx1, RANbp10, and Mgea5 as IDO2-binding proteins that do not interact with IDO1, implicating them as potential mediators of IDO2-specific function. Taken together, our findings identify a novel function for IDO2, independent of its tryptophan catabolizing activity, and suggest that this nonenzymatic function could involve multiple signaling pathways. These data show that the enzymatic activity of IDO2 is required only for some inflammatory immune responses and provide, to our knowledge, the first evidence of a nonenzymatic role for IDO2 in mediating autoimmune disease.

GAPDH inhibits intracellular pathways during starvation for cellular energy homeostasis.

Starvation poses a fundamental challenge to cell survival. Whereas the role of autophagy in promoting energy homeostasis in this setting has been extensively characterized1, other mechanisms are less well understood. Here we reveal that glyceraldehyde 3-phosphate dehydrogenase (GAPDH) inhibits coat protein I (COPI) transport by targeting a GTPase-activating protein (GAP) towards ADP-ribosylation factor 1 (ARF1) to suppress COPI vesicle fission. GAPDH inhibits multiple other transport pathways, also by targeting ARF GAPs. Further characterization suggests that this broad inhibition is activated by the cell during starvation to reduce energy consumption. These findings reveal a remarkable level of coordination among the intracellular transport pathways that underlies a critical mechanism of cellular energy homeostasis.

Glyceraldehyde-3-phosphate dehydrogenase regulates activation of c-Jun N-terminal kinase under oxidative stress.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) acts as a sensor under oxidative stress, leading to induction of various biological responses. Given that mitogen-activated protein kinase (MAPK) signaling pathways mediate cellular responses to a wide variety of stimuli, including oxidative stress, here, we aimed to elucidate whether a cross-talk cascade between GAPDH and MAPKs occurs under oxidative stress. Of the three typical MAPKs investigated-extracellular signal-regulated kinase, p38, and c-Jun N-terminal kinase (JNK)-we found that hydrogen peroxide (H2O2)-induced JNK activation is significantly reduced in HEK293 cells treated with small-interfering (si)RNA targeting GAPDH. Co-immunoprecipitation with a GAPDH antibody further revealed protein-protein interactions between GAPDH and JNK in H2O2-stmulated cells. Notably, both JNK activation and these interactions depend on oxidation of the active-site cysteine (Cys152) in GAPDH, as demonstrated by rescue experiments with either exogenous wild-type GAPDH or the cysteine-substituted mutant (C152A) in endogenous GAPDH-knockdown HEK293 cells. Moreover, H2O2-induced translocation of Bcl-2-associated X protein (Bax) into mitochondria, which occurs downstream of JNK activation, is attenuated by endogenous GAPDH knockdown in HEK293 cells. These results suggest a novel role for GAPDH in the JNK signaling pathway under oxidative stress.

Moonlighting glyceraldehyde-3-phosphate dehydrogenase: posttranslational modification, protein and nucleic acid interactions in normal cells and in human pathology.

Moonlighting glyceraldehyde-3-phosphate dehydrogenase (GAPDH) exhibits multiple functions separate and distinct from its historic role in energy production. Further, it exhibits dynamic changes in its subcellular localization which is an a priori requirement for its multiple activities. Separately, moonlighting GAPDH may function in the pathology of human disease, involved in tumorigenesis, diabetes, and age-related neurodegenerative disorders. It is suggested that moonlighting GAPDH function may be related to specific modifications of its protein structure as well as the formation of GAPDH protein: protein or GAPDH protein: nucleic acid complexes.

Determining the quantitative relationship between glycolysis and GAPDH in cancer cells exhibiting the Warburg effect.

Previous studies have identified GAPDH as a promising target for treating cancer and modulating immunity because its inhibition reduces glycolysis in cells (cancer cells and immune cells) with the Warburg effect, a modified form of cellular metabolism found in cancer cells. However, the quantitative relationship between GAPDH and the aerobic glycolysis remains unknown. Here, using siRNA-mediated knockdown of GAPDH expression and iodoacetate-dependent inhibition of enzyme activity, we examined the quantitative relationship between GAPDH activity and glycolysis rate. We found that glycolytic rates were unaffected by the reduction of GAPDH activity down to 19% ± 4.8% relative to untreated controls. However, further reduction of GAPDH activity below this level caused proportional reductions in the glycolysis rate. GAPDH knockdown or inhibition also simultaneously increased the concentration of glyceraldehyde 3-phosphate (GA3P, the substrate of GAPDH). This increased GA3P concentration countered the effect of GAPDH knockdown or inhibition and stabilized the glycolysis rate by promoting GAPDH activity. Mechanistically, the intracellular GA3P concentration is controlled by the Gibbs free energy of the reactions upstream of GAPDH. The thermodynamic state of the reactions along the glycolysis pathway was only affected when GAPDH activity was reduced below 19% ± 4.8%. Doing so moved the reactions catalyzed by GAPDH + PGK1 (phosphoglycerate kinase 1, the enzyme immediate downstream of GAPDH) away from the near-equilibrium state, revealing an important biochemical basis to interpret the rate control of glycolysis by GAPDH. Collectively, we resolved the numerical relationship between GAPDH and glycolysis in cancer cells with the Warburg effect and interpreted the underlying mechanism.

Glyceraldehyde-3-phosphate dehydrogenase present in extracellular vesicles from Leishmania major suppresses host TNF-alpha expression.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) fulfills various physiological roles that are unrelated to its glycolytic function. However, to date, the nonglycolytic function of GAPDH in trypanosomal parasites is absent from the literature. Exosomes secreted from Leishmania, like entire parasites, were found to have a significant impact on macrophage cell signaling and function, indicating cross talk with the host immune system. In this study, we demonstrate that the Leishmania GAPDH (LmGAPDH) protein is highly enriched within the extracellular vesicles (EVs) secreted during infection. To understand the function of LmGAPDH in EVs, we generated control, overexpressed, half-knockout (HKO), and complement cell lines. HKO cells displayed lower virulence compared with control cells when macrophages and BALB/c mice were infected with them, implying a crucial role for LmGAPDH in Leishmania infection and disease progression. Furthermore, upon infection of macrophages with HKO mutant Leishmania and its EVs, despite no differences in TNFA mRNA expression, there was a considerable increase in TNF-α protein expression compared with control, overexpressed, and complement parasites as determined by ELISA, RT-PCR, and immunoblot data. In vitro protein translation studies suggest that LmGAPDH-mediated TNF-α suppression occurs in a concentration-dependent manner. Moreover, mRNA binding assays also verified that LmGAPDH binds to the AU-rich 3'-UTR region of TNFA mRNA, limiting its production. Together, these findings confirmed that the LmGAPDH contained in EVs inhibits TNF-α expression in macrophages during infection via posttranscriptional repression.

Experimental evolution reveals a general role for the methyltransferase Hmt1 in noise buffering.

Cell-to-cell heterogeneity within an isogenic population has been observed in prokaryotic and eukaryotic cells. Such heterogeneity often manifests at the level of individual protein abundance and may have evolutionary benefits, especially for organisms in fluctuating environments. Although general features and the origins of cellular noise have been revealed, details of the molecular pathways underlying noise regulation remain elusive. Here, we used experimental evolution of Saccharomyces cerevisiae to select for mutations that increase reporter protein noise. By combining bulk segregant analysis and CRISPR/Cas9-based reconstitution, we identified the methyltransferase Hmt1 as a general regulator of noise buffering. Hmt1 methylation activity is critical for the evolved phenotype, and we also show that two of the Hmt1 methylation targets can suppress noise. Hmt1 functions as an environmental sensor to adjust noise levels in response to environmental cues. Moreover, Hmt1-mediated noise buffering is conserved in an evolutionarily distant yeast species, suggesting broad significance of noise regulation.

Glutathionylation primes soluble glyceraldehyde-3-phosphate dehydrogenase for late collapse into insoluble aggregates.

Protein aggregation is a complex physiological process, primarily determined by stress-related factors revealing the hidden aggregation propensity of proteins that otherwise are fully soluble. Here we report a mechanism by which glycolytic glyceraldehyde-3-phosphate dehydrogenase of Arabidopsis thaliana (AtGAPC1) is primed to form insoluble aggregates by the glutathionylation of its catalytic cysteine (Cys149). Following a lag phase, glutathionylated AtGAPC1 initiates a self-aggregation process resulting in the formation of branched chains of globular particles made of partially misfolded and totally inactive proteins. GSH molecules within AtGAPC1 active sites are suggested to provide the initial destabilizing signal. The following removal of glutathione by the formation of an intramolecular disulfide bond between Cys149 and Cys153 reinforces the aggregation process. Physiological reductases, thioredoxins and glutaredoxins, could not dissolve AtGAPC1 aggregates but could efficiently contrast their growth. Besides acting as a protective mechanism against overoxidation, S-glutathionylation of AtGAPC1 triggers an unexpected aggregation pathway with completely different and still unexplored physiological implications.

Evolved resistance to partial GAPDH inhibition results in loss of the Warburg effect and in a different state of glycolysis.

Aerobic glycolysis or the Warburg effect (WE) is characterized by increased glucose uptake and incomplete oxidation to lactate. Although the WE is ubiquitous, its biological role remains controversial, and whether glucose metabolism is functionally different during fully oxidative glycolysis or during the WE is unknown. To investigate this question, here we evolved resistance to koningic acid (KA), a natural product that specifically inhibits glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a rate-controlling glycolytic enzyme, during the WE. We found that KA-resistant cells lose the WE but continue to conduct glycolysis and surprisingly remain dependent on glucose as a carbon source and also on central carbon metabolism. Consequently, this altered state of glycolysis led to differential metabolic activity and requirements, including emergent activities in and dependences on fatty acid metabolism. These findings reveal that aerobic glycolysis is a process functionally distinct from conventional glucose metabolism and leads to distinct metabolic requirements and biological functions.

GAPDH delivers heme to soluble guanylyl cyclase.

Soluble guanylyl cyclase (sGC) is a key component of NO-cGMP signaling in mammals. Although heme must bind in the sGC ß1 subunit (sGCß) for sGC to function, how heme is delivered to sGCß remains unknown. Given that GAPDH displays properties of a heme chaperone for inducible NO synthase, here we investigated whether heme delivery to apo-sGCß involves GAPDH. We utilized an sGCß reporter construct, tetra-Cys sGCß, whose heme insertion can be followed by fluorescence quenching in live cells, assessed how lowering cell GAPDH expression impacts heme delivery, and examined whether expressing WT GAPDH or a GAPDH variant defective in heme binding recovers heme delivery. We also studied interaction between GAPDH and sGCß in cells and their complex formation and potential heme transfer using purified proteins. We found that heme delivery to apo-sGCß correlates with cellular GAPDH expression levels and depends on the ability of GAPDH to bind intracellular heme, that apo-sGCß associates with GAPDH in cells and dissociates when heme binds sGCß, and that the purified GAPDH-heme complex binds to apo-sGCß and transfers its heme to sGCß. On the basis of these results, we propose a model where GAPDH obtains mitochondrial heme and then forms a complex with apo-sGCß to accomplish heme delivery to sGCß. Our findings illuminate a critical step in sGC maturation and uncover an additional mechanism that regulates its activity in health and disease.

A nicotinamide phosphoribosyltransferase-GAPDH interaction sustains the stress-induced NMN/NAD+ salvage pathway in the nucleus.

All cells require sustained intracellular energy flux, which is driven by redox chemistry at the subcellular level. NAD+, its phosphorylated variant NAD(P)+, and its reduced forms NAD(P)/NAD(P)H are all redox cofactors with key roles in energy metabolism and are substrates for several NAD-consuming enzymes (e.g. poly(ADP-ribose) polymerases, sirtuins, and others). The nicotinamide salvage pathway, constituted by nicotinamide mononucleotide adenylyltransferase (NMNAT) and nicotinamide phosphoribosyltransferase (NAMPT), mainly replenishes NAD+ in eukaryotes. However, unlike NMNAT1, NAMPT is not known to be a nuclear protein, prompting the question of how the nuclear NAD+ pool is maintained and how it is replenished upon NAD+ consumption. In the present work, using human and murine cells; immunoprecipitation, pulldown, and surface plasmon resonance assays; and immunofluorescence, small-angle X-ray scattering, and MS-based analyses, we report that GAPDH and NAMPT form a stable complex that is essential for nuclear translocation of NAMPT. This translocation furnishes NMN to replenish NAD+ to compensate for the activation of NAD-consuming enzymes by stressful stimuli induced by exposure to H2O2 or S-nitrosoglutathione and DNA damage inducers. These results indicate that by forming a complex with GAPDH, NAMPT can translocate to the nucleus and thereby sustain the stress-induced NMN/NAD+ salvage pathway.

Glucose-dependent aerobic glycolysis contributes to recruiting viral components into HIV-1 particles to maintain infectivity.

The cellular environment affects optimal viral replication because viruses cannot replicate without their host cells. In particular, metabolic resources such as carbohydrates, lipids, and ATP are crucial for viral replication, which is sensitive to cellular metabolism. Intriguingly, recent studies have demonstrated that human immunodeficiency virus type 1 (HIV-1) infection induces a metabolic shift from oxidative phosphorylation to aerobic glycolysis in CD4+ T cells to produce the virus efficiently. However, the importance of aerobic glycolysis in maintaining the quality of viral components and viral infectivity has not yet been fully investigated. Here, we show that aerobic glycolysis is necessary not only to override the inhibitory effect of virion-incorporated glycolytic enzymes, but also to maintain the enzymatic activity of reverse transcriptase and the adequate packaging of envelope proteins into HIV-1 particles. To investigate the effect of metabolic remodeling on the phenotypic properties of HIV-1 produced by infected cells, we replaced glucose with galactose in the culture medium because the cells grown in galactose-containing medium are forced to carry out oxidative metabolism instead of aerobic glycolysis. We found that the packaging levels of glyceraldehyde 3-phosphate dehydrogenase, alpha-enolase and pyruvate kinase muscle type 2, which decrease HIV-1 infectivity by packaging into viral particles, are increased in progeny viruses produced by the cells grown in galactose-containing medium. Furthermore, we found that the entry and reverse transcription efficiency of the progeny viruses were reduced, which was caused by a decrease in the enzymatic activity of reverse transcriptase in the viral particles and a decrease in the packaging levels of envelope proteins and reverse transcriptase. These results indicate that the aerobic glycolysis environment in HIV-1-infected cells may contribute to the quality control of viruses.

Glycation of glyceraldehyde-3-phosphate dehydrogenase inhibits the binding with α-synuclein and RNA.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) shows great diversity of functions, interaction partners and post-translational modifications. GAPDH undergoes glycation of positively charged residues in diabetic patient's tissues and therefore may change interaction with partners. The influence of GAPDH glycation on interaction with two important partners, α-synuclein and RNA, has been investigated in silico using molecular dynamics simulations and in vitro using surface plasmon resonance measurements. Since positively charged groove including substrate- and NAD+-binding sites is proposed as potential binding site for α-synuclein and RNA, GAPDH was glycated on residues in grooves and randomly distributed over the whole surface. Lysine residues were replaced with negatively charged carboxymethyl lysine as a widespread advanced glycation end product. As results, GAPDH glycation suppressed the interaction with α-synuclein and RNA. Although the modified GAPDH residues participated in binding with α-synuclein, no stable binding site with both glycated forms was observed. Glycation along the whole GAPDH surface completely suppressed interaction with RNA, whereas the alternative possible RNA binding site was identified in case of groove glycation. The findings were supported by direct measurement of the binding affinity. The obtained results clarify effect of glycation on GAPDH interaction with α-synuclein and RNA and elucidate a possible mechanism of interplay between glycation occurred in diabetes and neurodegenerative diseases, which GAPDH and α-synuclein are involved in.

Src-mediated phosphorylation of GAPDH regulates its nuclear localization and cellular response to DNA damage.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key enzyme involved in energy metabolism. Recently, GAPDH has been suggested to have extraglycolytic functions in DNA repair, but the underlying mechanism for the GAPDH response to DNA damage remains unclear. Here, we demonstrate that the tyrosine kinase Src is activated under DNA damage stress and phosphorylates GAPDH at Tyr41. This phosphorylation of GAPDH is essential for its nuclear translocation and DNA repair function. Blocking the nuclear import of GAPDH by suppressing Src signaling or through a GAPDH Tyr41 mutation impairs its response to DNA damage. Nuclear GAPDH is recruited to DNA lesions and associates with DNA polymerase ß (Pol ß) to function in DNA repair. Nuclear GAPDH promotes Pol ß polymerase activity and increases base excision repair (BER) efficiency. Furthermore, GAPDH knockdown dramatically decreases BER efficiency and sensitizes cells to DNA damaging agents. Importantly, the knockdown of GAPDH in colon cancer SW480 cells and xenograft models effectively enhances their sensitivity to the chemotherapeutic drug 5-FU. In summary, our findings provide mechanistic insight into the new function of GAPDH in DNA repair and suggest a potential therapeutic target in chemotherapy.

Increased expression of GAPDH in cynomolgus monkeys with spontaneous cognitive decline and amyloidopathy reminiscent of an Alzheimer's-type disease is reflected in the circulation.

Previous studies of aging cynomolgus monkeys from our group identified spontaneous age-associated cognitive declines associated with biomarkers and brain lesions reminiscent of Alzheimer's Disease (AD), in a proportion of aged monkeys. However, the molecular mechanisms that underlie the spontaneous amyloid disorders and cognitive declines observed in these affected monkeys have yet to be investigated in detail. Using reverse transcriptase quantitative real time PCR techniques, normalized to the ACTB housekeeping gene, we analyzed the expression patterns of a number of genes which have been implicated in amyloid and tau abnormalities, in well-characterized aged cynomolgus monkeys with cognitive decline. A significantly increased expression of the genes coding for glyceraldehyde 3-phosphate dehydrogenase (GAPDH), was found in aged-cognitive decline monkeys compared to age-matched healthy controls. GAPDH has been implicated in several neurodegenerative diseases and interacts with beta amyloid precursor proteins. These findings provide support for the utilization of cynomolgus macaques in translational preclinical research as valid spontaneous models in experimental investigations of the relationships among aging, cognitive decline, and the neuropathy of AD.