Loss of O-GlcNAcase catalytic activity leads to defects in mouse embryogenesis.

O-GlcNAcylation is an essential post-translational modification that has been implicated in neurodevelopmental and neurodegenerative disorders. O-GlcNAcase (OGA), the sole enzyme catalyzing the removal of O-GlcNAc from proteins, has emerged as a potential drug target. OGA consists of an N-terminal OGA catalytic domain and a C-terminal pseudo histone acetyltransferase (HAT) domain with unknown function. To investigate phenotypes specific to loss of OGA catalytic activity and dissect the role of the HAT domain, we generated a constitutive knock-in mouse line, carrying a mutation of a catalytic aspartic acid to alanine. These mice showed perinatal lethality and abnormal embryonic growth with skewed Mendelian ratios after day E18.5. We observed tissue-specific changes in O-GlcNAc homeostasis regulation to compensate for loss of OGA activity. Using X-ray microcomputed tomography on late gestation embryos, we identified defects in the kidney, brain, liver, and stomach. Taken together, our data suggest that developmental defects during gestation may arise upon prolonged OGA inhibition specifically because of loss of OGA catalytic activity and independent of the function of the HAT domain.

Nutrient-driven O-GlcNAc in proteostasis and neurodegeneration.

Proteostasis is essential in the mammalian brain where post-mitotic cells must function for decades to maintain synaptic contacts and memory. The brain is dependent on glucose and other metabolites for proper function and is spared from metabolic deficits even during starvation. In this review, we outline how the nutrient-sensitive nucleocytoplasmic post-translational modification O-linked N-acetylglucosamine (O-GlcNAc) regulates protein homeostasis. The O-GlcNAc modification is highly abundant in the mammalian brain and has been linked to proteopathies, including neurodegenerative diseases such as Alzheimer's, Parkinson's, and Huntington's. C. elegans, Drosophila, and mouse models harboring O-GlcNAc transferase- and O-GlcNAcase-knockout alleles have helped define the role O-GlcNAc plays in development as well as age-associated neurodegenerative disease. These enzymes add and remove the single monosaccharide from protein serine and threonine residues, respectively. Blocking O-GlcNAc cycling is detrimental to mammalian brain development and interferes with neurogenesis, neural migration, and proteostasis. Findings in C. elegans and Drosophila model systems indicate that the dynamic turnover of O-GlcNAc is critical for maintaining levels of key transcriptional regulators responsible for neurodevelopment cell fate decisions. In addition, pathways of autophagy and proteasomal degradation depend on a transcriptional network that is also reliant on O-GlcNAc cycling. Like the quality control system in the endoplasmic reticulum which uses a 'mannose timer' to monitor protein folding, we propose that cytoplasmic proteostasis relies on an 'O-GlcNAc timer' to help regulate the lifetime and fate of nuclear and cytoplasmic proteins. O-GlcNAc-dependent developmental alterations impact metabolism and growth of the developing mouse embryo and persist into adulthood. Brain-selective knockout mouse models will be an important tool for understanding the role of O-GlcNAc in the physiology of the brain and its susceptibility to neurodegenerative injury.

O-GlcNAcylation stabilizes ß-catenin through direct competition with phosphorylation at threonine 41.

Dysfunctions in Wnt signaling increase ß-catenin stability and are associated with cancers, including colorectal cancer. In addition, ß-catenin degradation is decreased by nutrient-dependent O-GlcNAcylation. Human colon tumors and colons from mice fed high-carbohydrate diets exhibited higher amounts of ß-catenin and O-GlcNAc relative to healthy tissues and mice fed a standard diet, respectively. Administration of the O-GlcNAcase inhibitor thiamet G to mice also increased colonic expression of ß-catenin. By ETD-MS/MS, we identified 4 O-GlcNAcylation sites at the N terminus of ß-catenin (S23/T40/T41/T112). Furthermore, mutation of serine and threonine residues within the D box of ß-catenin reduced O-GlcNAcylation by 75%. Interestingly, elevating O-GlcNAcylation in human colon cell lines drastically reduced phosphorylation at T41, a key residue of the D box responsible for ß-catenin stability. Analyses of ß-catenin O-GlcNAcylation mutants reinforced T41 as the most crucial residue that controls the ß-catenin degradation rate. Finally, inhibiting O-GlcNAcylation decreased the ß-catenin/α-catenin interaction necessary for mucosa integrity, whereas O-GlcNAcase silencing improved this interaction. These results suggest that O-GlcNAcylation regulates not only the stability of ß-catenin, but also affects its localization at the level of adherens junctions. Accordingly, we propose that O-GlcNAcylation of ß-catenin is a missing link between the glucose metabolism deregulation observed in metabolic disorders and the development of cancer.

Unique hexosaminidase reduces metabolic survival signal and sensitizes cardiac myocytes to hypoxia/reoxygenation injury.

Metabolic signaling through the posttranslational linkage of N-acetylglucosamine (O-GlcNAc) to cellular proteins represents a unique signaling paradigm operative during lethal cellular stress and a pathway that we and others have recently shown to exert cytoprotective effects in vitro and in vivo. Accordingly, the present work addresses the contribution of the hexosaminidase responsible for removing O-GlcNAc (ie, O-GlcNAcase) from proteins. We used pharmacological inhibition, viral overexpression, and RNA interference of O-GlcNAcase in isolated cardiac myocytes to establish its role during acute hypoxia/reoxygenation. Elevated O-GlcNAcase expression significantly reduced O-GlcNAc levels and augmented posthypoxic cell death. Conversely, short interfering RNA directed against, or pharmacological inhibition of, O-GlcNAcase significantly augmented O-GlcNAc levels and reduced posthypoxic cell death. On the mechanistic front, we evaluated posthypoxic mitochondrial membrane potential and found that repression of O-GlcNAcase activity improves, whereas augmentation impairs, mitochondrial membrane potential recovery. Similar beneficial effects on posthypoxic calcium overload were also evident. Such changes were evident without significant alteration in expression of the major putative components of the mitochondrial permeability transition pore (ie, voltage-dependent anion channel, adenine nucleotide translocase, cyclophilin D). The present results provide definitive evidence that O-GlcNAcase antagonizes posthypoxic cardiac myocyte survival. Moreover, such results support a renewed approach to the contribution of metabolism and metabolic signaling to the determination of cell fate.

Ym1/2 promotes Th2 cytokine expression by inhibiting 12/15(S)-lipoxygenase: identification of a novel pathway for regulating allergic inflammation.

The Ym1/2 lectin is expressed abundantly in the allergic mouse lung in an IL-13-dependent manner. However, the role of Ym1/2 in the development of allergic airways disease is largely unknown. In this investigation, we show that treatment of mice with anti-Ym1/2 Ab during induction of allergic airways disease attenuated mediastinal lymph node production of IL-5 and IL-13. Ym1/2 was found to be expressed by dendritic cells (DCs) in an IL-13-dependent manner and supplementation of DC/CD4(+) T cell cocultures with Ym1/2 enhanced the ability of IL-13(-/-) DCs to stimulate the secretion of IL-5 and IL-13. Affinity chromatography identified 12/15(S)-lipoxygenase (12/15-LOX) as a Ym1/2-interacting protein and functional studies suggested that Ym1/2 promoted the ability of DCs to stimulate cytokine production by inhibiting 12/15-LOX-mediated catalysis of 12-hydroxyeicosatetraenoic acid (12(S)-HETE). Treatment of DC/CD4(+) T cell cultures with the 12/15-LOX inhibitor baicalein enhanced, whereas 12(S)-HETE inhibited the production of Th2 cytokines. Notably, delivery of 12(S)-HETE to the airways of mice significantly attenuated the development of allergic airways inflammation and the production of IL-5 and IL-13. In summary, our results suggest that production of Ym1/2 in response to IL-13 promotes Th2 cytokine production and allergic airways inflammation by inhibiting the production of 12(S)-HETE by 12/15-LOX.

Role for lysosomal enzyme beta-hexosaminidase in the control of mycobacteria infection.

The pathogenic mycobacteria that cause tuberculosis (TB) and TB-like diseases in humans and animals elude sterilizing immunity by residing within an intracellular niche in host macrophages, where they are protected from microbicidal attack. Recent studies have emphasized microbial mechanisms for evasion of host defense; less is known about mycobactericidal mechanisms that remain intact during initial infection. To better understand macrophage mechanisms for restricting mycobacteria growth, we examined Mycobacterium marinum infection of Drosophila S2 cells. Among approximately 1,000 host genes examined by RNAi depletion, the lysosomal enzyme beta-hexosaminidase was identified as an important factor in the control of mycobacterial infection. The importance of beta-hexosaminidase for restricting mycobacterial growth during mammalian infections was confirmed in macrophages from beta-hexosaminidase knockout mice. Beta-hexosaminidase was characterized as a peptidoglycan hydrolase that surprisingly exerts its mycobactericidal effect at the macrophage plasma membrane during mycobacteria-induced secretion of lysosomes. Thus, secretion of lysosomal enzymes is a mycobactericidal mechanism that may have a more general role in host defense.

Activation of the XBP1s/O-GlcNAcylation Pathway Improves Functional Outcome After Cardiac Arrest and Resuscitation in Young and Aged Mice.

ABSTRACT: After cardiac arrest (CA) and resuscitation, the unfolded protein response (UPR) is activated in various organs including the brain. However, the role of the UPR in CA outcome remains largely unknown. One UPR branch involves spliced X-box-binding protein-1 (XBP1s). Notably, XBP1s, a transcriptional factor, can upregulate expression of specific enzymes related to glucose metabolism, and subsequently boost O-linked ß-N-acetylglucosamine modification (O-GlcNAcylation). The current study is focused on effects of the XBP1 UPR branch and its downstream O-GlcNAcylation on CA outcome. Using both loss-of-function and gain-of-function mouse genetic tools, we provide the first evidence that activation of the XBP1 UPR branch in the post-CA brain is neuroprotective. Specifically, neuron-specific Xbp1 knockout mice had worse CA outcome, while mice with neuron-specific expression of Xbp1s in the brain had better CA outcome. Since it has been shown that the protective role of the XBP1s signaling pathway under ischemic conditions is mediated by increasing O-GlcNAcylation, we then treated young mice with glucosamine, and found that functional deficits were mitigated on day 3 post CA. Finally, after confirming that glucosamine can boost O-GlcNAcylation in the aged brain, we subjected aged mice to 8 min CA, and then treated them with glucosamine. We found that glucosamine-treated aged mice performed significantly better in behavioral tests. Together, our data indicate that the XBP1s/O-GlcNAc pathway is a promising target for CA therapy.

A potent mechanism-inspired O-GlcNAcase inhibitor that blocks phosphorylation of tau in vivo.

Pathological hyperphosphorylation of the microtubule-associated protein tau is characteristic of Alzheimer's disease (AD) and the associated tauopathies. The reciprocal relationship between phosphorylation and O-GlcNAc modification of tau and reductions in O-GlcNAc levels on tau in AD brain offers motivation for the generation of potent and selective inhibitors that can effectively enhance O-GlcNAc in vertebrate brain. We describe the rational design and synthesis of such an inhibitor (thiamet-G, K(i) = 21 nM; 1) of human O-GlcNAcase. Thiamet-G decreased phosphorylation of tau in PC-12 cells at pathologically relevant sites including Thr231 and Ser396. Thiamet-G also efficiently reduced phosphorylation of tau at Thr231, Ser396 and Ser422 in both rat cortex and hippocampus, which reveals the rapid and dynamic relationship between O-GlcNAc and phosphorylation of tau in vivo. We anticipate that thiamet-G will find wide use in probing the functional role of O-GlcNAc in vertebrate brain, and it may also offer a route to blocking pathological hyperphosphorylation of tau in AD.

Chronic hyperglycemia induces tau hyperphosphorylation by downregulating OGT-involved O-GlcNAcylation in vivo and in vitro.

OBJECTIVE: Diabetes mellitus (DM) can increase the risk of cognitive dysfunction, but its exact mechanisms remain unclear. The involvement of aberrant O-GlcNAcylation has been identified in hyperglycemia and DM, as well as the pathogenesis of Alzheimer's disease via competition with tau phosphorylation. This study was designed to investigate the role of O-GlcNAcylation in diabetes-associated cognitive dysfunction (DACD). METHODS: Fifteen-week old male KK-Ay mice were used as DACD models, and advanced glycation end product (AGE)-treated HT22 cells were used as a model of high glucose toxicity. Morris water maze tests, histological staining, real-time quantitative PCR, and Western blot were also applied. RESULTS: Mice with DACD exhibited evident obesity, hyperinsulinemia, hyperglycemia, and impaired learning and memory function. O-GlcNAcylation levels decreased and tau phosphorylation levels at Ser396, Ser404, Thr212, and Thr231 increased in the hippocampus of mice with DACD, as well as in AGE-treated HT22 cells. Hypoglycemic therapy improved these anomalies and elevated O-GlcNAc transferase (OGT) levels in mice with DACD. OGT plasmid transfection in HT22 cells partially reversed AGE-induced decreases in O-GlcNAcylation levels and increased tau phosphorylation levels. CONCLUSIONS: Chronic hyperglycemia can induce tau hyperphosphorylation by downregulating OGT-involved O-GlcNAcylation in vivo and in vitro, which mediates DACD.

Microglia are continuously activated in the circumventricular organs of mouse brain.

Microglia are the primary resident immune cells of the brain parenchyma and transform into the amoeboid form in the "activated state" under pathological conditions from the ramified form in the "resting state" under physiologically healthy conditions. In the present study, we found that microglia in the circumventricular organs (CVOs) of adult mice displayed the amoeboid form with fewer branched cellular processes even under normal conditions; however, those in other brain regions showed the ramified form, which is characterized by well-branched and dendritic cellular processes. Moreover, microglia in the CVOs showed the strong protein expression of the M1 markers CD16/32 and CD86 and M2 markers CD206 and Ym1 without any pathological stimulation. Thus, the present results indicate that microglia in the CVOs of adult mice are morphologically and functionally activated under normal conditions, possibly due to the specialized features of the CVOs, namely, the entry of blood-derived molecules into parenchyma through fenestrated capillaries and the presence of neural stem cells.

Roles of pgaABCD genes in synthesis, modification, and export of the Escherichia coli biofilm adhesin poly-beta-1,6-N-acetyl-D-glucosamine.

The linear homopolymer poly-beta-1,6-N-acetyl-D-glucosamine (beta-1,6-GlcNAc; PGA) serves as an adhesin for the maintenance of biofilm structural stability in diverse eubacteria. Its function in Escherichia coli K-12 requires the gene products of the pgaABCD operon, all of which are necessary for biofilm formation. PgaC is an apparent glycosyltransferase that is required for PGA synthesis. Using a monoclonal antibody directed against E. coli PGA, we now demonstrate that PgaD is also needed for PGA formation. The deletion of genes for the predicted outer membrane proteins PgaA and PgaB did not prevent PGA synthesis but did block its export, as shown by the results of immunoelectron microscopy (IEM) and antibody adsorption assays. IEM also revealed a conditional localization of PGA at the cell poles, the initial attachment site for biofilm formation. PgaA contains a predicted beta-barrel porin and a superhelical domain containing tetratricopeptide repeats, which may mediate protein-protein interactions, implying that it forms the outer membrane secretin for PGA. PgaB contains predicted carbohydrate binding and polysaccharide N-deacetylase domains. The overexpression of pgaB increased the primary amine content (glucosamine) of PGA. Site-directed mutations targeting the N-deacetylase catalytic activity of PgaB blocked PGA export and biofilm formation, implying that N-deacetylation promotes PGA export through the PgaA porin. The results of previous studies indicated that N-deacetylation of beta-1,6-GlcNAc in Staphylococcus epidermidis by the PgaB homolog, IcaB, anchors it to the cell surface. The deletion of icaB resulted in release of beta-1,6-GlcNAc into the growth medium. Thus, covalent modification of beta-1,6-GlcNAc by N-deacetylation serves distinct biological functions in gram-negative and gram-positive species, dictated by cell envelope differences.

Revisiting glycoside hydrolase family 20 ß-N-acetyl-d-hexosaminidases: Crystal structures, physiological substrates and specific inhibitors.

Glycoside hydrolase family 20 ß-N-acetyl-d-hexosaminidases (GH20s) catalyze the hydrolysis of glycosidic linkages in glycans, glycoproteins and glycolipids. The diverse substrates of GH20s account for their various roles in many important bioprocesses, such as glycoprotein modification, glycoconjugate metabolism, gamete recognition and chitin degradation in fungal cell walls and arthropod exoskeletons. Defects in human GH20s cause lysosomal storage diseases, Alzheimer's disease and osteoarthritis. Similarly, lower levels of GH20s arrest arthropod molting. Although GH20s are promising targets for drug and agrochemical development, designing bioactive molecules to target one specific enzyme is challenging because GH20s share a conserved catalytic mechanism. With the development of structural biology, the last two decades have witnessed a dramatic increase in crystallographic investigations of liganded and unliganded GH20s, providing core information for rational molecular designs. This critical review summarizes recent research advances in GH20s, with a focus on their structural basis of substrate specificity as well as on inhibitor design. As more crystal structures of targeted GH20s are determined and analyzed, dynamics of their catalysis and inhibition will also be elucidated, which will facilitate the development of new drugs, pesticides and agrochemicals.

A genetic model of substrate deprivation therapy for a glycosphingolipid storage disorder.

Inherited defects in the degradation of glycosphingolipids (GSLs) cause a group of severe diseases known as GSL storage disorders. There are currently no effective treatments for the majority of these disorders. We have explored a new treatment paradigm, substrate deprivation therapy, by constructing a genetic model in mice. Sandhoff's disease mice, which abnormally accumulate GSLs, were bred with mice that were blocked in their synthesis of GSLs. The mice with simultaneous defects in GSL synthesis and degradation no longer accumulated GSLs, had improved neurologic function, and had a much longer life span. However, these mice eventually developed a late-onset neurologic disease because of accumulation of another class of substrate, oligosaccharides. The results support the validity of the substrate deprivation therapy and also highlight some limitations.

The cranial base in craniofacial development: a gene therapy study.

The etiology of midface retrusion remains largely unclear. We hypothesized that the cranial base synchondroses play a key role in the development of the craniofacial skeleton in the Sandhoff mouse model. We observed that developmental abnormalities of the cranial base synchondroses involving proliferative chondrocytes are important in craniofacial growth and development. Neonatal restitution of beta-hexosaminidase in mutant mice by gene therapy successfully ameliorated the attendant skeletal defects and restored craniofacial morphology in vivo, suggesting this as a critical temporal window in craniofacial development. Analysis of our data implicates parathyroid-related peptide (PTHrP) and cyclo-oxygenase-2 (COX-2) as possible factors underlying the development of the aforementioned skeletal defects. Hence, timely restitution of a genetic deficiency or, alternatively, the restoration of PTHrP or cyclo-oxygenase activity by the administration of PTH and/or non-steroidal anti-inflammatory drugs or COX-2 selective inhibitors to affected individuals may prove beneficial in the management of midface retrusion.

Effects of exogenous N-acetylhexosaminidase on the structure and mineralization of the post-ecdysial exoskeleton of the blue crab, Callinectes sapidus.

A cuticular glycosidase with characteristics of N-acetyl-beta-D-hexosaminidase (HexNAcase) was identified in post-ecdysial crab cuticle. Its appearance coincided with changes in cuticular glycoproteins and the onset of mineralization. To test if HexNAcase might be the causative agent in the alteration of the glycans and initiation of calcification, newly molted crab cuticle was treated with exogenous HexNAcase. Treating cuticular extracts from crabs at 0 h post-ecdysis with exogenous HexNAcase mimicked those changes observed in vivo. Specifically, the enzyme decreased the concanavalin A affinity of an 83-kDa glycoprotein that binds to calcite crystals in vitro. Treating pieces of 0 h post-ecdysial cuticle with HexNAcase rendered them capable of nucleating calcite in vitro (similar to 5 h post-ecdysial cuticle), while untreated, 0 h controls remained uncalcified. The data imply a role of the cuticular HexNAcase-like enzyme in the initiation of calcite nucleation in the newly formed exoskeleton.

Serum hexosaminidase and beta-glucuronidase activities in infants: effects of age and sex.

We investigated the effect of age and sex on the serum activity of hexosaminidase (HEX) and -glucuronidase (BGLU) in 275 normal term infants aged 12 h to 12 months. Up to six weeks of life, HEX was significantly higher in boys (P<=0.023). During the age period of 1-26 weeks, BGLU was also higher in boys, but differences were significant only at 2-6 and 7-15 weeks (P<=0.016). The developmental pattern of HEX and BGLU was sex dependent. HEX activity increased in both sexes from 4-7 days of life, reaching a maximum of 1.4-fold the birth value at 2-6 weeks of age in boys (P<0.001) and a maximum of 1.6-fold at 7-15 weeks in girls (P<0.001). HEX activity gradually decreased thereafter, reaching significantly lower levels at 27-53 weeks than during the first three days of life in boys (P = 0.002) and the same level of this age interval in girls. BGLU increased in both sexes from 4-7 days of age, showing a maximum increase at 7-15 weeks (3.3-fold in boys and 2.9-fold in girls, both P<0.001). Then BGLU decreased in boys to a value similar to that observed at 4-7 days of age. In girls, BGLU remained elevated until the end of the first year of life. These results indicate a variation of HEX and BGLU activities during the first year of life and a sex influence on their developmental pattern. This observation should be considered in the diagnosis of GM2 gangliosidosis and mucopolysaccharidosis type VII.

miR128 up-regulation correlates with impaired amyloid ß(1-42) degradation in monocytes from patients with sporadic Alzheimer's disease.

Alzheimer's disease (AD), the most common form of dementia in elderly individuals, is characterized by neurofibrillary tangles, extracellular amyloid-ß (Aß) plaques and neuroinflammation. New evidence has shown that the lysosomal system might be a crossroad in which etiological factors in AD pathogenesis converge. This study shows that several lysosomal enzymes, including Cathepsin B, D, S, ß-Galactosidase, α-Mannosidase, and ß-Hexosaminidase, were less expressed in monocytes and lymphocytes from patients with a clinical diagnosis of AD dementia compared with cells from healthy controls. In vitro experiments of gain and loss of function suggest that down-regulation is a direct consequence of miR-128 up-regulation found in AD-related cells. The present study also demonstrates that miR-128 inhibition in monocytes from AD patients improves Aß(1-42) degradation. These results could contribute to clarify the molecular mechanisms that affect the imbalanced Aß production/clearance involved in the pathogenesis of AD.

Identification of glucocorticoid-regulated genes that control cell proliferation during murine respiratory development.

Glucocorticoids play a vital role in fetal respiratory development and act via the intracellular glucocorticoid receptor (GR) to regulate transcription of key target genes. GR-null mice die at birth due to respiratory dysfunction associated with hypercellularity and atelectasis. To identify events associated with this lung phenotype we examined perinatal cellular proliferation rates and apoptotic indices. We demonstrate that compared to wild-type controls, day 18.5 postcoitum (p.c.) GR-null mouse lungs display significantly increased cell proliferation rates (1.8-fold P < 0.05) and no change in apoptosis. To examine underlying molecular mechanisms, we compared whole genome expression profiles by microarray analysis at 18.5 days p.c. Pathways relating to cell proliferation, division and cell cycle were significantly down-regulated while pathways relating to carbohydrate metabolism, kinase activities and immune responses were significantly up-regulated. Differential levels of gene expression were verified by quantitative-RT-PCR and/or Northern analysis. Key regulators of proliferation differentially expressed in the lung of 18.5 p.c. GR-null lungs included p21 CIP1 (decreased 2.9-fold, P < 0.05), a negative regulator of the cell cycle, and Mdk (increased 6.0-fold, P < 0.05), a lung growth factor. The more under-expressed genes in 18.5 p.c. GR-null lungs included Chi3l3 (11-fold, P < 0.05), a macrophage inflammatory response gene and Ela1 (9.4-fold, P < 0.05), an extracellular matrix remodeling enzyme. Our results demonstrate that GR affects the transcriptional status of a number of regulatory processes during late fetal lung development. Amongst these processes is cell proliferation whereby GR induces expression of cell cycle repressors while suppressing induction of a well characterized cell cycle stimulator.