Sladjana Dukic-Stefanovic

Advanced glycation endproducts co-localize with inducible nitric oxide synthase in Alzheimer’s disease

Advanced glycation endproducts (AGEs), protein-bound oxidation products of sugars, have been shown to be involved in the pathophysiological processes of Alzheimers disease (AD). AGEs induce the expression of various pro-inflammatory cytokines and the inducible nitric oxide synthase (iNOS) leading to a state of oxidative stress. AGE modification and resulting crosslinking of protein deposits such as amyloid plaques may contribute to the oxidative stress occurring in AD. The aim of this study was to immunohistochemically compare the localization of AGEs and beta-amyloid (Abeta) with iNOS in the temporal cortex (Area 22) of normal and AD brains. In aged normal individuals as well as early stage AD brains (i.e. no pathological findings in isocortical areas), a few astrocytes showed co-localization of AGE and iNOS in the upper neuronal layers, compared with no astrocytes detected in young controls. In late AD brains, there was a much denser accumulation of astrocytes co-localized with AGE and iNOS in the deeper and particularly upper neuronal layers. Also, numerous neurons with diffuse AGE but not iNOS reactivity and some AGE and iNOS-positive microglia were demonstrated, compared with only a few AGE-reactive neurons and no microglia in controls. Finally, astrocytes co-localized with AGE and iNOS as well as AGE and were found surrounding mature but not diffuse amyloid plaques in the AD brain. Our results show that AGE-positive astrocytes and microglia in the AD brain express iNOS and support the evidence of an AGE-induced oxidative stress occurring in the vicinity of the characteristic lesions of AD. Hence activation of microglia and astrocytes by AGEs with subsequent oxidative stress and cytokine release may be an important progression factor in AD.

Anti-inflammatory antioxidants attenuate the expression of inducible nitric oxide synthase mediated by advanced glycation endproducts in murine microglia

Advanced glycation endproducts (AGEs) accumulate on long‐lived protein deposits including β‐amyloid plaques in Alzheimers disease (AD). AGE‐modified amyloid deposits contain oxidized and nitrated proteins as markers of a chronic neuroinflammatory condition and are surrounded by activated microglial and astroglial cells. We show in this study that AGEs increase nitric oxide production by induction of the inducible nitric oxide synthase (iNOS) on the mRNA and protein level in the murine microglial cell line N‐11. Membrane permeable antioxidants including oestrogen derivatives (e.g. 17β‐oestradiol) thiol antioxidants (e.g. (R+)‐α‐lipoic acid) and Gingko biloba extract EGb 761, but not phosphodiesterase inhibitors such as propentophylline, prevent the up‐regulation of AGE‐induced iNOS expression and NO production. These results indicate that oxygen free radicals serve as second messengers in AGE‐induced pro‐inflammatory signal transduction pathways. As this pharmacological mechanism is not only relevant for Alzheimers disease, but also for many chronic inflammatory conditions, such membrane‐permeable antioxidants could be regarded not only as antioxidant, but also as potent therapeutic anti‐inflammatory drugs.

β‐Amyloid peptide potentiates inflammatory responses induced by lipopolysaccharide, interferon ‐γ and ‘advanced glycation endproducts’ in a murine microglia cell line

β‐Amyloid (Aβ) plaques are characteristic hallmarks of Alzheimers disease (AD). In AD, it has been suggested that activation of microglial cells might be the link between Aβ deposition and neuronal degeneration. Activated microglia are associated with senile plaques and produce free radicals and inflammatory cytokines. However, it is still not clear whether Aβ needs a prestimulated environment to exert its proinflammatory potential. Advanced glycation endproducts (AGEs), protein‐bound oxidation products of sugars, have been shown to accumulate in senile plaques and could induce a silent but chronic inflammation in the AD brain. We tested whether Aβ acts as an amplifier of a submaximal proinflammatory response initiated by exposure to chicken egg albumin‐AGE, lipopolysaccharide or interferon‐γ. Synthetic Aβ was used to produce three different samples (Aβ‐fibrilar; Aβ‐aggregated; Aβ‐AGE), which were characterized for β‐sheeted fibrils by the thioflavin‐T test and electron microscopy. As markers of microglial activation, nitric oxide, interleukin‐6, macrophage‐colony stimulation factor and tumour necrosis factor‐α production was measured. All three Aβ samples alone could not induce a detectable microglial response. The combination of Aβ preparations, however, with the coinducers provoked a strong microglial response, whereby Aβ‐AGE and fibrilar Aβ were more potent inflammatory signals than aggregated Aβ. Thus, Aβ in senile plaques can amplify microglial activation by a coexisting submaximal inflammatory stimulus. Hence, anti‐inflammatory therapeutics could either target the primary proinflammatory signal (e.g. by limiting AGE‐formation by AGE inhibitors or cross‐link breakers) or the amplifyer Aβ (e.g. by limiting Aβ production by β‐ or γ‐secretase inhibitors).

Microglial activation induces cell death, inhibits neurite outgrowth and causes neurite retraction of differentiated neuroblastoma cells

Abstract.Activation of glial cells has been proposed to contribute to neuronal dysfunction and neuronal cell death in Alzheimers disease. In this study, we attempt to determine some of the effects of secreted factors from activated murine N-11 microglia on viability and morphology of neurons using the differentiated neuroblastoma cell line Neuro2a. Microglia were activated either by lipopolysaccharide (LPS), bacterial cell wall proteoglycans, or advanced glycation endproducts (AGEs), protein-bound sugar oxidation products. At high LPS or AGE concentrations, conditioned medium from microglia caused neuronal cell death in a dose-dependent manner. At sublethal LPS or AGE concentrations, conditioned media inhibited retinoic acid-induced neurite outgrowth and stimulated retraction of already extended neurites. Among the many possible secreted factors, the contribution of NO or NO metabolites in the cytotoxicity of conditioned medium was investigated. Cell death and changes in neurite morphology were partly reduced when NO production was inhibited by nitric oxide synthase inhibitors. The results suggest that even in the absence of significant cell death, inflammatory processes, which are partly transmitted via NO metabolites, may affect intrinsic functions of neurons such as neurite extension that are essential components of neuronal morphology and thus may contribute to degenerative changes in Alzheimers disease.

Signal transduction pathways in mouse microglia N-11 cells activated by advanced glycation endproducts (AGEs)

Deposition of cross‐linked insoluble protein aggregates such as amyloid plaques is characteristic for Alzheimers disease. Microglial activation by these extracullar deposits has been proposed to play a crucial role in functional degeneration as well as cell death of neurones. A sugar‐derived post‐translational modification of long‐lived proteins, advanced glycation endproducts (AGEs), activate specific signal transduction pathways, resulting in the up‐regulation of various pro‐inflammatory signals such as cytokines [interleukin‐6 (IL‐6), tumour necrosis factor‐alpha (TNF‐α)] and inducible nitric oxide synthase (iNOS). Our goal was to study AGE‐activated signal transduction pathways involved in the induction of pro‐inflammatory effectors in the murine microglial cell line N‐11. Chicken egg albumin‐AGE (CEA‐AGE), used as model AGE, induces nitric oxide (NO), TNF‐α and IL‐6 production. The AGE receptor, RAGE, and the transcription factor, nuclear factor kappa B (NF‐κB), appear to be involved in all pathways, since a neutralizing RAGE antibody and a peptide inhibiting NF‐κB translocation down‐regulated NO, TNF‐α and IL‐6 production. NO and TNF‐α, but not IL‐6 production appear to be regulated independently, since NOS inhibitors did not decrease TNF‐α secretion and a neutralizing TNF‐α antibody did not reduce NO production, while employment of NOS inhibitors reduced significantly the secretion of IL‐6. Inhibition of the MAP‐kinase‐kinase (MEK) and phosphatidylinositol 3‐kinase (PI3K) pathway, but not that of mitogen‐activated protein kinase‐p38 (MAPK‐p38), reduced NO, TNF‐α and IL‐6 significantly, suggesting that simultaneous activation of the first two pathways is necessary for the AGE‐induced induction of these pro‐inflammatory stimuli.

Expression of Aquaporins in the Retina of Diabetic Rats

Purpose/Aim: The development of retinal edema is the main reason of impaired vision in non-proliferative diabetic retinopathy. Water transport through aquaporins (AQPs) has been suggested to facilitate the development of ischemic edema in the retina. Here, we investigated whether experimental diabetic retinopathy in rats results in alterations of the AQP expression in the neural retina and retinal pigment epithelium (RPE). Materials and Methods: Experimental diabetes in rats was induced by a single intravenous injection of streptozotocin (65 mg/kg body weight). The gene expression of AQPs in tissues from control and diabetic rats was examined by real-time RT-PCR. Retinal cryosections were immunostained against AQP5, 6, and 9. Results: The total RNAs extracted from the neural retina and RPE contained gene transcripts for AQP0, 1, 3, 4, 5, 6, 8, 9, 11, and 12. Experimental diabetes was associated with an upregulation of AQP1 in the neural retina, and of AQP5, 9, 11, and 12 in the RPE. Furthermore, diabetes was associated with a downregulation of AQP6 and AQP11 in the neural retina, and of AQP0 in the RPE. AQP5 and AQP9 immunolabelings of the RPE were increased, and AQP6 labeling of the outer plexiform layer was decreased in retinal slices from diabetic rats in comparison to slices from control rats. Conclusions: The data suggest that experimental diabetic retinopathy is associated with a complex pattern of alteration in the retinal AQP expression. These alterations might be involved in the adaptation of retinal cells to hyperglycemic conditions and the development and/or resolution of retinal edema.

Immunolocalization of aquaporin-6 in the rat retina

Previous RT-PCR experiments revealed that the neural retina of the rat contains gene transcripts of numerous aquaporins (AQPs), including AQP6 (Tenckhoff et al., Neuroreport 16 (2005) 53-56). In the present study, we investigated the localization of AQP6 immunoreactivity in slices of the rat neural retina, and determined whether blue light injury of the retina affects the tissue distribution of this channel. AQP6 immunoreactivity was found to be selectively localized to the outer plexiform layer. Around the ribbon synapses in this layer, AQP6 labeling was co-localized with the glial water channel AQP4. AQP6 labeling was not colocalized with the marker of horizontal cells, calbindin, nor with the marker of rod bipolar cells, protein kinase Cα. Along with the degeneration of photoreceptor cells after blue light treatment of the retina, AQP6-labeled ribbon synapses disappeared, and a punctate AQP6 staining redistributed into the inner nuclear layer. The co-localization of AQP6 and the glial water channel AQP4 suggests a preferential localization of AQP6 in glial membranes that surround the ribbon synapses in the outer plexiform layer. AQP6 might be involved in the glia-mediated osmo and ion regulation of the extracellular space in this layer.

Chorionic gonadotropin and its receptor are both expressed in human retina, possible implications in normal and pathological conditions.

Extra-gonadal role of gonadotropins has been re-evaluated over the last 20 years. In addition to pituitary secretion of luteinizing hormone (LH) and follicle stimulating hormone (FSH), the CNS has been clearly identified as a source of hCG acting locally to influence behaviour. Here we demonstrated that human retina is producing this gonadotropin that acts as a neuroactive molecule. Müller glial and retinal pigmented epithelial (RPE) cells are producing hCG that may affects neighbour cells expressing its receptor, namely cone photoreceptors. It was previously described that amacrine and retinal ganglion (RGC) cells are targets of the gonadotropin releasing hormone that control the secretion of all gonadotropins. Therefore our findings suggest that a complex neuroendocrine circuit exists in the retina, involving hCG secreting cells (glial and RPE), hCG targets (photoreceptors) and hCG-release controlling cells (amacrine and RGC). The exact physiological functions of this circuit have still to be identified, but the proliferation of photoreceptor-derived tumor induced by hCG demonstrated the need to control this neuroendocrine loop.

Efficient Photodynamic Therapy on Human Retinoblastoma Cell Lines

Photodynamic therapy (PDT) has shown to be a promising technique to treat various forms of malignant neoplasia. The photodynamic eradication of the tumor cells is achieved by applying a photosensitizer either locally or systemically and following local activation through irradiation of the tumor mass with light of a specific wavelength after a certain time of incubation. Due to preferential accumulation of the photosensitizer in tumor cells, this procedure allows a selective inactivation of the malignant tumor while sparing the surrounding tissue to the greatest extent. These features and requirements make the PDT an attractive therapeutic option for the treatment of retinoblastoma, especially when surgical enucleation is a curative option. This extreme solution is still in use in case of tumours that are resistant to conventional chemotherapy or handled too late due to poor access to medical care in less advanced country. In this study we initially conducted in-vitro investigations of the new cationic water-soluble photo sensitizer tetrahydroporphyrin-tetratosylat (THPTS) regarding its photodynamic effect on human Rb-1 and Y79 retinoblastoma cells. We were able to show, that neither the incubation with THPTS without following illumination, nor the sole illumination showed a considerable effect on the proliferation of the retinoblastoma cells, whereas the incubation with THPTS combined with following illumination led to a maximal cytotoxic effect on the tumor cells. Moreover the phototoxicity was lower in normal primary cells from retinal pigmented epithelium demonstrating a higher phototoxic effect of THPTS in cancer cells than in this normal retinal cell type. The results at hand form an encouraging foundation for further in-vivo studies on the therapeutic potential of this promising photosensitizer for the eyeball and vision preserving as well as potentially curative therapy of retinoblastoma.

Development of a Novel Nonpeptidic 18F-Labeled Radiotracer for in Vivo Imaging of Oxytocin Receptors with Positron Emission Tomography

With the aim of imaging and quantification of oxytocin receptors (OTRs) in living brain using positron emission tomography (PET), we developed a (18)F-labeled small molecule radiotracer and investigated its in vivo pharmacokinetics in mice and pig. [(18)F]6b (KD = 12.3 nM) was radiolabeled by a two-step procedure using a microwave system with radiochemical yields of 26.9 ± 4.7%. Both organ distribution and small animal PET studies revealed limited brain uptake of [(18)F]6b in mouse (mean SUV of 0.04 at 30 min pi). Besides, significant radioactivity uptake in the pituitary gland was observed (SUV of 0.7 at 30 min pi). In a dynamic PET study in one piglet, we detected a higher uptake of [(18)F]6b in the olfactory bulb (SUV of 0.34 at 30 min pi) accompanied by a low uptake in the whole brain. In vitro autoradiographic studies on porcine brain sections indicated interaction of [(18)F]6b with several off-target receptors.