Marina Hermes

Contribution of E-NTPDase1 (CD39) to renal protection from ischemia-reperfusion injury

Previous studies showed increased extracellular nucleotides during renal ischemia‐reperfusion. While nucleotides represent the main source for extracellular adenosine and adenosine signaling contributes to renal protection from ischemia, we hypothesized a role for ecto‐nucleoside‐triphosphate‐diphosphohydro‐lases (E‐NTPDases) in renal protection. We used a model of murine ischemia‐reperfusion and in situ ischemic preconditioning (IP) via a hanging weight system for atraumatic renal artery occlusion. Initial studies with a nonspecific inhibitor of E‐NTPDases (POM‐1) revealed inhibition of renal protection by IP. We next pursued transcriptional responses of E‐NTPDases (E‐NTPDasel‐3, and 8) to renal IP, and found a robust and selective induction of E‐NTPDase1/CD39 transcript and protein. Moreover, based on clearance studies, plasma electrolytes, and renal tubular histology, IP protection was abolished in gene‐targeted mice for cd39 whereas increased renal adenosine content with IP was attenuated. Furthermore, administration of apyrase reconstituted renal protection by IP in cd39−/− mice. Finally, apyrase treatment of wild‐type mice resulted in increased renal adenosine concentrations and a similar degree of renal protection from ischemia as IP treatment. Taken together, these data identify CD39‐dependent nucleotide phosphohydrolysis in renal protection. Moreover, the present studies suggest apyrase treatment as a novel pharmacological approach to renal diseases precipitated by limited oxygen availability.—Grenz, A., Zhang, H., Hermes, M., Eckle, T., Klingel, K., Huang, D. Y., Muller, C. E., Robson, S. C., Osswald, H., Eltzschig, H. K. Contribution of E‐NTPDasel (CD39) to renal protection from ischemia‐reperfusion injury. FASEB J. 21, 2863–2873 (2007)

Dermcidin-Derived Peptides Show a Different Mode of Action than the Cathelicidin LL-37 against Staphylococcus aureus

ABSTRACT Dermcidin (DCD) is an antimicrobial peptide which is constitutively expressed in eccrine sweat glands. By postsecretory proteolytic processing in sweat, the DCD protein gives rise to anionic and cationic DCD peptides with a broad spectrum of antimicrobial activity. Many antimicrobial peptides induce membrane permeabilization as part of their killing mechanism, which is accompanied by a loss of the bacterial membrane potential. In this study we show that there is a time-dependent bactericidal activity of anionic and cationic DCD-derived peptides which is followed by bacterial membrane depolarization. However, DCD-derived peptides do not induce pore formation in the membranes of gram-negative and gram-positive bacteria. This is in contrast to the mode of action of the cathelicidin LL-37. Interestingly, LL-37 as well as DCD-derived peptides inhibit bacterial macromolecular synthesis, especially RNA and protein synthesis, without binding to microbial DNA or RNA. Binding studies with components of the cell envelope of gram-positive and gram-negative bacteria and with model membranes indicated that DCD-derived peptides bind to the bacterial envelope but show only a weak binding to lipopolysaccharide (LPS) from gram-negative bacteria or to peptidoglycan, lipoteichoic acid, and wall teichoic acid, isolated from Staphylococcus aureus. In contrast, LL-37 binds strongly in a dose-dependent fashion to these components. Altogether, these data indicate that the mode of action of DCD-derived peptides is different from that of the cathelicidin LL-37 and that components of the bacterial cell envelope play a role in the antimicrobial activity of DCD.

Intracellular calcium signalling in Alzheimer’s disease

•  Introduction •  Dysregulation of Ca2+ homeostasis in AD ‐  Aβ accumulation causes Ca2+ dyshomeostasis ‐  Ca2+ dyshomeostasis increases Aβ production •  Presenilins and Ca2+ homeostasis •  Dysregulation of Ca2+ homeostasis in vivo •  AD‐mediated hyperactivity and synaptic network dysfunction •  Neuronal hyperactivity: implications for humans •  Plaque vicinity •  Conclusions

S-Adenosylhomocysteine Metabolism in Different Cell Lines: Effect of Hypoxia and Cell Density

Background/Aims: The methylation potential (MP) is defined as the ratio of S-adenosylmethionine (AdoMet) to S-adenosylhomocysteine (AdoHcy). It was shown recently that hypoxia increases AdoMet/AdoHcy ratio in HepG2 cells (Hermes et al., Exp Cell Res 294: 325-334, 2004). In the present study, we compared AdoMet/AdoHcy ratio and energy metabolism in HepG2, HEK-293, HeLa, MCF-7 and SK-HEP-1 cell lines under normoxia and hypoxia. Methods: Metabolite concentrations were measured by HPLC. In addition, AdoHcy hydrolase (AdoHcyase) activity was determined photometrically. Results: Under normoxia HepG2 cells show the highest AdoMet/AdoHcy ratio of 53.4 ± 3.3 followed by MCF-7 and SK-HEP-1 cells with a AdoMet/AdoHcy ratio of 14.4 ± 1.1 and 21.1 ± 1.3, respectively. The lowest AdoMet/AdoHcy ratios are exhibited by HeLa and HEK-293 cells (6.6 ± 0.7 and 7.1 ± 0.3). Hypoxia does not significantly change the MP in MCF-7 and HeLa cells, but alters the MP in HepG2, HEK-293 and SK-HEP-1 cells. These alterations are dependent on the cell density. Under normoxia HepG2 cells exhibit AdoHcyase activity of 2.5 ± 0.2 nmol min-1 mg-1 protein. All other cell lines show 3-5 times lower enzyme activity. Interestingly, hypoxia affects AdoHcyase activity only in HepG2 cells. Conclusions: Our data clearly show that the cell lines are characterized by different MP and different behavior under hypoxia. That implies that a lower MP is not necessarily associated with impaired transmethylation activity and cellular function.

Expression and Localization of S-Adenosylhomocysteine-Hydrolase in the Rat Kidney Following Carbon Monoxide Induced Hypoxia

Background/Aims: Tissue hypoxia induces a variety of functional changes including enhanced transcriptional activity associated with high transmethylation activity (e.g. mRNA cap methylation) in the nucleus. It is well known that the kidney responds to hypoxia with enhanced transcription of erythropoietin (EPO) in the interstitial cells. Since S-adenosylhomocysteine (AdoHcy)-hydrolase regulates most S-adenosylmethionine (AdoMet) dependent transmethylation reactions by hydrolyzing the potent feedback inhibitor AdoHcy to adenosine and homocysteine we studied the effect of hypoxia by carbon monoxide (CO) inhalation (1200ppm) on AdoHcy-hydrolase gene expression and its localization in rat kidneys. Results: CO lowered renal AdoHcy-hydrolase mRNA expression by 64% whereas AdoHcy-hydrolase activity was not changed during 4h of CO exposure 0.7±0.04mU/mg (control) vs. 0.75±0.06mU/mg protein. Using two-channel immunofluorescence confocal laser scanning microscope AdoHcy-hydrolase was visualized in different cells of the hypoxic rat kidney. A very bright immunofluorescence of AdoHcy-hydrolase was observed in the nuclei of single interstitial cells of renal cortex and outer medulla which respond to hypoxia with increased EPO secretion indicating translocation of AdoHcy-hydrolase from the cytosol to the nucleus. Conclusions: These data suggest that AdoHcy-hydrolase accumulation in the nucleus of adult mammalian cells is involved in maintaining efficient transmethylation reactions in transcriptionally active cells by removing the product inhibitor AdoHcy.

High-resolution in vivo imaging of microglia using a versatile nongenetically encoded marker

Microglial cells are the innate immune cells of the CNS, whose main role is to monitor the integrity of and to react to any disturbances of brain homeostasis. As such, microglial cells are involved in a large number of CNS insults (e.g. acute CNS injury, brain tumors, apoptosis, infection, ischemia, neurodegenerative diseases) and their engagement can be either neurotoxic or neuroprotective [1,2]. Despite their critical role in ameliorating or exacerbating disease progression, little is known about the in vivo functional properties of these cells. So far high resolution in vivo studies of microglial function were conducted in mice with genetically labeled microglia [3,4]. These analyses revealed the surveillance function of microglia in the healthy brain [5,6], and its involvement in the remodeling of synaptic circuits during ischemia and sensory deprivation [7]. However, because of the lack of fractalkine receptor (CX3CR1 mice) or low expression levels of GFP (Iba1-GFP mice), these mouse lines are less suitable for studying the role of microglia under pathological conditions [2,8–10]. Therefore, there is a need for a nongenetically encoded, easy to use microglial marker, enabling highquality staining of microglia (similar to the quality obtained in GFP-expressing mice), but applicable to any mouse strain at any experimental age. Here we utilize a well-known histological marker tomato lectin (from Lycopersicon esculentum [11–13]) for high resolution in vivo imaging of microglia. A brief pressure injection of tomato lectin conjugated with a fluorescent dye DyLight R

In vivo odourant response properties of migrating adult-born neurons in the mouse olfactory bulb

Juxtaglomerular neurons (JGNs) of the mammalian olfactory bulb are generated throughout life. Their integration into the preexisting neural network, their differentiation and survival therein depend on sensory activity, but when and how these adult-born cells acquire responsiveness to sensory stimuli remains unknown. In vivo two-photon imaging of retrovirally labelled adult-born JGNs reveals that ~90% of the cells arrive at the glomerular layer after day post injection (DPI) 7. After arrival, adult-born JGNs are still migrating, but at DPI 9, 52% of them have odour-evoked Ca(2+) signals. Their odourant sensitivity closely resembles that of the parent glomerulus and surrounding JGNs, and their spontaneous and odour-evoked spiking is similar to that of their resident neighbours. Our data reveal a remarkably rapid functional integration of adult-born cells into the preexisting neural network. The mature pattern of odour-evoked responses of these cells strongly contrasts with their molecular phenotype, which is typical of immature, migrating neuroblasts.

S-Adenosylhomocysteine hydrolase overexpression in HEK-293 cells: effect on intracellular adenosine levels, cell viability, and DNA methylation.

Background/Aims: S-Adenosylhomocysteine hydrolase (AdoHcyase) catalyzes the reversible hydrolysis of S-adenosylhomocysteine (AdoHcy), which is a potent product inhibitor of S-adenosylmethionine (AdoMet)-dependent methyltransferases. While previous studies have shown that AdoHcyase inhibition or deficiency lead to a decreased AdoMet/AdoHcy ratio resulting in impaired transmethylation, the effect of enhanced AdoHcyase activity on AdoMet/AdoHcy metabolism and methylation reactions has not been studied in detail. Methods: To investigate the effect of enhanced AdoHcyase activity, we generated HEK-293 cell lines stably overexpressing AdoHcyase. Results: Initial studies revealed that 2-10-fold AdoHcyase overexpression resulted in decreased intracellular AdoHcy and elevated adenosine levels, whereas 16-fold AdoHcyase overexpression increased adenosine and AdoHcy levels, lowered energy charge, and altered cell morphology. Furthermore, we found a correlation between AdoHcyase activity and cell viability. Caspase-activity assays and DNA fragmentation analysis revealed that the cell death in AdoHcyase overexpressing cells was due to apoptosis. Global DNA methylation was not altered in the different AdoHcyase overexpressing cell lines. Conclusion: Taken together, these data show that 2-5-fold enhanced AdoHcyase activity is well tolerated by the cell, while greatly enhanced AdoHcyase activity results in adenosine-induced apoptosis. The fact that enhanced AdoHcyase activity does not increase transmethylation activity suggests that AdoHcyase activity under physiological conditions is not rate limiting for efficient transmethylation.

A bell-shaped dependence between amyloidosis and GABA accumulation in astrocytes in a mouse model of Alzheimer's disease

Functioning at the interface between the nervous and immune systems, in the amyloid-depositing brain, astrocytes become hypertrophic and accumulate around senile plaques. Moreover, hippocampal astrocytes upregulate their γ-aminobutyric acid (GABA) content and enhance tonic inhibition, likely causing local circuit imbalance. It remains, however, unclear whether this effect is hippocampus specific and how it is regulated during disease progression. Here, we studied changes in astrocytic morphology and GABA content in the frontal cortex and dentate gyrus of control and amyloid-depositing mice. Healthy aging was accompanied by a transient increase in astrocytic GABA content at middle age and region-specific alterations of soma size. In contrast, amyloid deposition caused a gradual cortex-accentuated increase in soma size. Importantly, our data uncovered a bell-shaped relationship between the mouse age and astrocytic GABA content in both brain regions. Moreover, in mice carrying an Alzheimers disease-related mutation in presenilin 1, astrocytes accumulated GABA even in the absence of amyloidosis. These data question the proposed inhibition of astrocytic GABA synthesis as a universal strategy for treating network dysfunction in Alzheimers disease.

Hyperhomocysteinemia is Associated with Decreased Erythropoietin Expression in Rats

Background/Aims: Elevated plasma homocysteine (Hcy) levels have been identified as a pathogenic factor causing a variety of pathological changes in different cells and tissues. In vertebrates, Hcy is produced solely from S-adenosylhomocysteine (AdoHcy) through the catalysis of AdoHcy-hydrolase. The direction of AdoHcy-hydrolase activity is determined by its cytosolic substrate concentrations, thereby controlling intracellular AdoHcy levels. Most S-adenosylmethionine (AdoMet)-dependent methyltransferases are regulated in vivo by the ratio of AdoMet/AdoHcy, which is termed “methylation potential” (MP). To test whether high rates of erythropoietin (EPO) expression is reduced by a low MP in vivo we choosed the model of increased EPO production following carbon monoxide (CO) exposure in rats in which high transcriptional activity is responsible for renal EPO production. Results: To induce a sustained hyperhomocysteinemia in rats, we infused i.v. a low or high dose of Hcy resulting in Hcy plasma levels of 87.4±6.2 and 300.8±23.7 µmol/l, respectively. Renal tissue contents of AdoHcy, AdoMet, and adenosine (Ado) were measured after freeze clamp by means of HPLC. Within 4h of CO exposure EPO serum levels increased from 13.6±0.4 (control) to 2254.8±278.3 mIU/ml. Only high dose of Hcy reduces both, the MP from 40.8±2.0 to 8.2±1.0 in the kidney as well as EPO serum levels by 40% compared to control rats. Conclusion: Our data show that severe hyperhomocysteinemia (HHcy) affects the MP in the renal tissue and lowers EPO expression following CO induced intoxication. This result supports the concept that efficient EPO production requires an unimpaired MP.