Incorporation of N-propanoylneuraminic acid leads to calcium oscillations in oligodendrocytes upon the application of GABA

AbstractSialylation of glycoproteins and glycolipids plays an important role during development, regeneration and pathogenesis. It has been shown that unnatural sialylation within glial cell cultures can have distinct effects on their proliferation and antigenic profiles. These cultures metabolize N-propanoylmannosamine (N-propanoylneuraminic acid precursor=P-NAP), a synthetic non-physiological precursor of neuraminic acid, resulting in the expression of N-propanoylneuraminic acid in glycoconjugates of their cell membranes [Schmidt, C., Stehling, P., Schnitzer, J., Reutter, W. and Horstkorte, R. (1998) J. Biol. Chem. 273, 19146–19152]. To determine whether these biochemically engineered sialic acids influence calcium concentrations in cells of the oligodendrocyte lineage, mixed glial cultures of oligodendrocytes growing on top of an astrocyte monolayer were exposed to glutamate, histamine, adrenaline, γ-aminobutyric acid (GABA), high potassium (high K+) and ATP. Calcium responses in P-NAP-treated oligodendrocytes were determined by confocal microscopy with the calcium indicator fluo-3 AM, and compared with control cultures. We showed that P-NAP differentially modulated the calcium responses of individual oligodendrocytes when GABA was applied. GABA induced calcium oscillations with up to four spikes per min in 60% of oligodendrocytes when treated with P-NAP

Glycation Interferes with the Activity of the Bi-Functional UDP-N-Acetylglucosamine 2-Epimerase/N-Acetyl-mannosamine Kinase (GNE)

Mutations in the gene coding for the bi-functional UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE), the key enzyme of the sialic acid biosynthesis, are responsible for autosomal-recessive GNE myopathy (GNEM). GNEM is an adult-onset disease with a yet unknown exact pathophysiology. Since the protein appears to work adequately for a certain period of time even though the mutation is already present, other effects appear to influence the onset and progression of the disease. In this study, we want to investigate whether the late onset of GNEM is based on an age-related effect, e.g., the accumulation of post-translational modifications (PTMs). Furthermore, we also want to investigate what effect on the enzyme activity such an accumulation would have. We will particularly focus on glycation, which is a PTM through non-enzymatic reactions between the carbonyl groups (e.g., of methylglyoxal (MGO) or glyoxal (GO)) with amino groups of proteins or other biomolecules. It is already known that the levels of both MGO and GO increase with age. For our investigations, we express each domain of the GNE separately, treat them with one of the glycation agents, and determine their activity. We demonstrate that the enzymatic activity of the N-acetylmannosamine kinase (GNE-kinase domain) decreases dramatically after glycation with MGO or GO—with a remaining activity of 13% ± 5% (5 mM MGO) and 22% ± 4% (5 mM GO). Whereas the activity of the UDP-N-acetylglucosamine 2-epimerase (GNE-epimerase domain) is only slightly reduced after glycation—with a remaining activity of 60% ± 8% (5 mM MGO) and 63% ± 5% (5 mM GO).Deutsche Forschungsgemeinschaft (DFG, German Research Foundation)Deutsche ForschungsgemeinschaftPeer Reviewe

The homozygous M712T mutation of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase results in reduced enzyme activities but not in altered overall cellular sialylation in hereditary inclusion body myopathy

AbstractHereditary inclusion body myopathy (HIBM) is a neuromuscular disorder, caused by mutations in UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase, the key enzyme of sialic acid biosynthesis. In Middle Eastern patients a single homozygous mutation occurs, converting methionine-712 to threonine. Recombinant expression of the mutated enzyme revealed slightly reduced N-acetylmannosamine kinase activity, in agreement with the localization of the mutation within the kinase domain. B lymphoblastoid cell lines derived from patients expressing the mutated enzyme also display reduced UDP-N-acetylglucosamine 2-epimerase activity. Nevertheless, no reduced cellular sialylation was found in those cells by colorimetric assays and lectin analysis, indicating that HIBM is not directly caused by an altered overall expression of sialic acids

Biochemical engineering of the N-acyl side chain of sialic acid leads to increased calcium influx from intracellular compartments and promotes differentiation of HL60 cells

AbstractSialylation of glycoconjugates is essential for mammalian cells. Sialic acid is synthesized in the cytosol from N-acetylmannosamine by several consecutive steps. Using N-propanoylmannosamine, a novel precursor of sialic acid, we are able to incorporate unnatural sialic acids with a prolonged N-acyl side chain (e.g., N-propanoylneuraminic acid) into glycoconjugates taking advance of the cellular sialylation machinery. Here, we report that unnatural sialylation of HL60-cells leads to an increased release of intracellular calcium after application of thapsigargin, an inhibitor of SERCA Ca2+-ATPases. Furthermore, this increased intracellular calcium concentration leads to an increased adhesion to fibronectin. Finally, we observed an increase of the lectin galectin-3, a marker of monocytic differentiation of HL60-cells

Wnt Glycation Inhibits Canonical Signaling

Glycation occurs as a non-enzymatic reaction between amino and thiol groups of proteins, lipids, and nucleotides with reducing sugars or α-dicarbonyl metabolites. The chemical reaction underlying is the Maillard reaction leading to the formation of a heterogeneous group of compounds named advanced glycation end products (AGEs). Deleterious effects have been observed to accompany glycation such as alterations of protein structure and function resulting in crosslinking and accumulation of insoluble protein aggregates. A substantial body of evidence associates glycation with aging. Wnt signaling plays a fundamental role in stem cell biology as well as in regeneration and repair mechanisms. Emerging evidence implicates that changes in Wnt/β-catenin pathway activity contribute to the aging process. Here, we investigated the effect of glycation of Wnt3a on its signaling activity. Methods: Glycation was induced by treatment of Wnt3a-conditioned medium (CM) with glyoxal (GO). Effects on Wnt3a signaling activity were analyzed by Topflash/Fopflash reporter gene assay, co-immunoprecipitation, and quantitative RT-PCR. Results: Our data show that GO-treatment results in glycation of Wnt3a. Glycated Wnt3a suppresses β-catenin transcriptional activity in reporter gene assays, reduced binding of β-catenin to T-cell factor 4 (TCF-4) and extenuated transcription of Wnt/β-catenin target genes. Conclusions: GO-induced glycation impairs Wnt3a signaling function

Mutation of a putative MAP kinase consensus site regulates NCAM endocytosis and NCAM-dependent neurite outgrowth

International audienceThe cytoplasmic domain of the neural cell adhesion molecule NCAM contains several putative serine/threonine phosphorylation sites whose functions are largely unknown. Human NCAM140 (NCAM140) possesses a potential MAP kinase phosphorylation site at threonine (T) 803. The aim of this study was to analyze a possible phosphorylation of NCAM140 by MAP kinases and to identify the functional role of T803. We found that NCAM140 is phosphorylated by the MAP kinase ERK2 in vitro. Exchange of T803 to aspartic acid (D) which mimics constitutive phosphorylation at the respective position resulted in increased endocytosis compared to NCAM140 in neuroblastoma cells and primary neurons. Consistently, NCAM140 endocytosis was inhibited by the MEK inhibitor U0126 in contrast to NCAM140-T803D or NCAM140-T803A endocytosis supporting a role of a potential ERK2 mediated phosphorylation at this site in endocytosis. Furthermore, cells expressing NCAM140-T803D developed significantly shorter neurites than NCAM140 expressing cells indicating that a potential phosphorylation of NCAM by ERK2 also regulates NCAM-dependent neurite outgrowth

Investigation of the Neuroprotective Impact of Nimodipine on Neuro2a Cells by Means of a Surgery-Like Stress Model

Nimodipine is well characterized for the management of SAH (subarachnoid hemorrhage) and has been shown to promote a better outcome and less DIND (delayed ischemic neurological deficits). In rat experiments, enhanced axonal sprouting and higher survival of motoneurons was demonstrated after cutting or crushing the facial nerve by nimodipine. These results were confirmed in clinical trials following vestibular Schwannoma surgery. The mechanism of the protective competence of nimodipine is unknown. Therefore, in this study, we established an in vitro model to examine the survival of Neuro2a cells after different stress stimuli occurring during surgery with or without nimodipine. Nimodipine significantly decreased ethanol-induced cell death of cells up to approximately 9% in all tested concentrations. Heat-induced cell death was diminished by approximately 2.5% by nimodipine. Cell death induced by mechanical treatment was reduced up to 15% by nimodipine. Our findings indicate that nimodipine rescues Neuro2a cells faintly, but significantly, from ethanol-, heat- and mechanically-induced cell death to different extents in a dosage-dependent manner. This model seems suitable for further investigation of the molecular mechanisms involved in the neuroprotective signal pathways influenced by nimodipine

Disturbance of Key Cellular Subproteomes upon Propofol Treatment Is Associated with Increased Permeability of the Blood-Brain Barrier

Background: Propofol is a short-acting anesthetic, which is often used for induction and maintenance of general anesthesia, sedation for mechanically ventilated adults and procedural sedation. Several side effects of propofol are known and a substantial number of patients suffer from post-operative delirium after propofol application. In this study, we analyzed the effect of propofol on the function and protein expression profile on a proteome-wide scale. Methods: We cultured human brain microvascular endothelial cells in absence and presence of propofol and analyzed the permeability of the blood-brain barrier (BBB) by fluorescein passage and protein abundance on a proteome-wide scale by mass spectrometry. Results: Propofol interfered with the function of the blood-brain barrier. This was not due to decreased adhesion of propofol-treated human brain microvascular endothelial cells. The proteomic analysis revealed that some key pathways in these cells were disturbed, such as oxygen metabolism, DNA damage recognition and response to stress. Conclusions: Propofol has strong effects on protein expression which could explain several side effects of propofol