Symposium Biologie
Freitag, 09.09.2016
10:45 - 12:15
Studio A
ProMoAge: Proteinmodification and Ageing

Moderation: L. Klotz, Jena; A. Simm, Halle (Saale)

Ageing leads to impairment of tissue homeostasis and functional decline of organs and represents a main risk factor for prevalent diseases such as cardiovascular disease and neurodegeneration in developed countries. To devise therapies aimed at improving the health state of the elderly, a detailed knowledge of molecular mechanisms leading to the impairment of organ function with increasing age is essential. There is accumulating evidence that posttranslational modifications (PTMs) of proteins contribute to this decline. PTMs occur either enzymatically catalysed or non-enzymatically, with both types often targeting the same amino acid. Under physiological conditions enzymatic PTMs regulate protein activities, thereby controlling shape and function of cells. However, due to alterations of modifying enzymes or because of an altered cellular environment in aged organisms, PTM patterns may change, and, moreover, non-enzymatic modifications, such as oxidation or glycation, may compete with enzymatically regulated processes, such as acetylation and glycosylation. As a consequence, dysregulation of cellular processes occurs. The session will focus on such PTMs and their role in aging.

Novel insights in the dysfunction of human blood-brain barrier after glycation

K. Bork, M. Hussein, R. Horstkorte; Halle (Saale)

Formation of advanced glycation endproducts (AGE) are discussed to play a role in ageing and also in impaired BBB function. Advanced glycation endproducts represent posttranslational modifications generated by irreversible non-enzymatic crosslinking reactions between carbonyl-containing molecules (e.g. sugars) and amino groups of proteins-a reaction referred to as glycation. Glycation is one feature of ageing and leads to the formation of non-degradable and less functional proteins and enzymes and can additionally induce inflammation and further pathophysiological processes. The blood-brain barrier (BBB) provides a dynamic and complex interface consisting of endothelial cells, pericytes and astrocytes, which are embedded in a collagen and fibronectin-rich basement membrane. This complex structure restricts the diffusion of small hydrophilic solutes and macromolecules as well as the transmigration of leukocytes into the brain. Age related dysfunction of the BBB are associated with the progressive deterioration of central nervous system (CNS) function. It has been shown that carbonyl stress followed by the formation of AGEs interfere with the BBB integrity and function. Here, we present data that carbonyl stress induced by methylglyoxal leads to glycation of endothelial cells and the basement membrane, which interferes with the barrier-function and with the expression of RAGE, occludin and ZO-1. Furthermore, methylglyoxal induced carbonyl stress promotes the expression of the pro-inflammatory interleukins IL-6 and IL-8. In summary, this study provides new insights into the relationship between AGE formation by carbonyl stress and brain microvascular endothelial barrier dysfunction.

Does the maternal metabolism during early pregnancy determine the offspring´s health and ageing?

A. Navarrete Santos, M. Schindler, T. Seeling, E. Haucke, A. Navarrete Santos, K. Grybel, M. Pendzialek, J. Gürke, R. Heller, A. Simm, B. Fischer; Halle (Saale), Jena

According to the thesis of “Developmental origins of health and diseases” (DOHAD) changes in embryonic cell physiology program later development and predispose for metabolic diseases and ageing. The preimplantation period is a critical ontogenetic stage in embryo development and highly vulnerable for teratogenesis and epigenetic modifications. At this period the embryo is most sensitive to its surrounding milieu, especially to deregulations by external stimuli.
In particular the embryogenesis of the rabbit is a suitable model for humans to perform molecular studies on preimplantation embryos and the influence of maternal diseases on embryo development. As in women a diabetes mellitus type 1 during the peri-conceptional period leads to subfertility with metabolic and hormonal changes in the reproductive organs of female rabbits. As a consequence, the preimplantation embryo adapt to the diabetic uterine environment by global changes in metabolism. In diabetic rabbits an oversupply of maternal nutrients as glucose, amino acids and lipids in uterine secretions, correlates with a non-physiological high accumulation of lipids and enforced protein synthesis in the embryo. A conversion of embryonic metabolism from insulin- to adiponectin-dependent glucose metabolism allows the embryo to adapt to a diabetic environment. Maternal hyperglycemia during early pregnancy was correlated with an increase in AGE formation in the uterine environment and the embryo itself. AGE accumulation and the activation of RAGE influenced the development of the embryo by increasing AGE-mediated cellular stress. The embryos from diabetic rabbits showed a higher ROS production and protein modifications, indicating that embryos were metabolically imbalanced. We suppose that the high plasticity of embryonic cells and embryonic stress adaptation are potential targets for programming of metabolic and age-related diseases like diabetes mellitus in adulthood.

Supported by the German Research Council (DFG ProMoAge, GRK 2155) and EU (Epihealth, EpihealthNET)

AMPK effects angiogenesis via regulation of O-GlcNAcylation in endothelial cells

D. Zibrova, F. Vandermoere, O. Göransson, M. Peggie, A. Knierim, K. Spengler, B. Viollet, N. A. Morrice, K. Sakamoto, R. Heller; Jena, Dundee/GB, Lund/S, Paris/F

Aims: Adenosine monophosphate-activated protein kinase (AMPK), a master sensor of the cellular energy levels contributes to an anti-inflammatory and anti-atherogenic phenotype of endothelial cells as well as to the regulation of angiogenesis. However, the molecular mechanisms mediating these functions remain elusive. We identified glutamine:fructose-6-phosphate amidotransferase 1 (GFAT1) as a potential AMPK substrate with serine 243 as a phosphorylation site in mouse embryonic fibroblasts. GFAT1 is the rate-limiting enzyme of the hexosamine biosynthesis pathway (HBP), which converts glucose to UDP-N-acetylglucosamine (UDP-GlcNAc). The latter mediates posttranslational modification (O-GlcNAcylation) of various cellular proteins. The current study was aimed at characterizing GFAT1 as an AMPK substrate in endothelial cells and a possible functional role of the AMPK-GFAT axis in angiogenesis. Material and methods: Experiments were performed in human umbilical vein endothelial cells (HUVEC), in which AMPK was downregulated by specific siRNAs or stimulated with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) or vascular endothelial growth factor (VEGF). The phosphorylation states of AMPK and its substrates were determined by immunoblotting of total lysates or immunoprecipitates. Total O-GlcNAc levels were analysed by immunoblotting using an anti-O-GlcNAc antibody. To investigate the involvement of HBP in angiogenesis, sprouting of capillary-like structures from the endothelial cell spheroids was evaluated in the presence or absence of the GFAT antagonist 6-diazo-5-oxonorleucine (DON) or after downregulation of GFAT1 by siRNA. To verify the role of AMPK-mediated GFAT1 phosphorylation at serine 243, a HUVEC cell line was generated, in which the non-phosphorylatable S243A-GFAT1 mutant was overexpressed.
Results: The chemical AMPK activator AICAR and VEGF, a major physiological AMPK agonist, increased GFAT1 phosphorylation in primary endothelial cells but not when the AMPKα catalytic subunits were downregulated by siRNAs. O-GlcNAcylation was considerably increased in HUVEC with AMPKα double knockdown. Oppositely, O-GlcNAc levels were decreased in cells treated with AICAR but the inhibitory effect of AICAR on protein O-GlcNAcylation was low in HUVEC expressing the S243A-GFAT1 mutant. Together these data suggest that AMPK inhibits GFAT1 activity by phosphorylating its serine residue 243. Pharmacological inhibition or downregulation of GFAT1 increased basal and VEGF-induced angiogenesis indicating that the HBP has an antiangiogenic role which may be counteracted by AMPK. Indeed, when the inhibitory phosphorylation by AMPK was prevented by introduction of the S243A-GFAT1 mutant, VEGF-induced angiogenesis was decreased.
Conclusions: The present study verifies GFAT1 as an AMPK substrate in endothelial cells. By phosphorylating GFAT1 at serine 243, AMPK decreases O-GlcNAc levels of endothelial proteins and thereby regulates angiogenesis in response to the physiological stimulus VEGF. Our findings provide novel insights into the role of HBP in angiogenesis and suggest that targeting AMPK in endothelium might improve disturbed angiogenesis in conditions of high HBP activity as seen in hyperglycemia. The AMPK/GFAT1 axis represents a novel angiogenic pathway activated by VEGF.

Cellular thiol status is a determinant of stress resistance and life span in C. elegans

N. Urban, D. Tsitsipatis, K. Kreuzer, F. Hausig, H. Steinbrenner, L.-O. Klotz; Jena

Cellular thiols – both protein and low-molecular mass (lmm) thiols – constitute a major line of defense against oxidative damage. We here tested how exposure to thiol-depleting agents affects stress signaling, stress resistance and life span of C. elegans worms. Depletion of lmm thiols using a high concentration of diethyl maleate (DEM, 1 mM) caused acute stimulation of stress signaling in the exposed worms, resulting in nuclear accumulation of the transcription factor DAF-16. Despite the well-known role of DAF-16 in extending C. elegans life span, viability of worms exposed to 1 mM DEM was dramatically lowered, whilst gene expression of DAF-16 target genes was not up-regulated. However, growing worms in the presence of DEM at lower concentrations (10-100 µM) resulted in an extension of mean nematode life span by approx. 10%. These worms were significantly more resistant against exposure to the redox cycling herbicide paraquat than control worms. Interestingly, no acute nuclear accumulation of DAF-16 was observed under these conditions. DEM-induced life span extension was not detectable in worms with inactivated DAF-16, pointing to a role of DAF-16 in the effect despite the non-detectable nuclear accumulation. Furthermore, inactivation of SKN-1, another stress-responsive transcription factor controlling the expression of cytoprotective genes, had the same effect, pointing to a collaborative role of DAF-16 and SKN-1 in enhancing C. elegans life span in worms exposed to low DEM concentrations. In line with these findings for lower DEM concentrations, the expression of DAF-16 and SKN-1 target genes was elevated under these conditions. In order to test whether glutathione (GSH) is the lmm thiol responsible for the observed effects, we depleted GSH by downregulating γ-glutamylcysteine synthetase (GCS), the enzyme catalyzing the rate-limiting step in GSH biosynthesis. Depletion started at young adult stage and allowed for C. elegans survival. Moderate decrease of GSH due to knockdown of GCS did not impair viability but rather significantly elevated life span. In summary, modest GSH depletion – through pharmacologic or genetic approaches – may elevate C. elegans life span, and DAF-16 and SKN-1 contribute to thiol depletion-induced life span extension.