Annette Maczurek

Inflammation and the Redox‐sensitive AGE–RAGE Pathway as a Therapeutic Target in Alzheimer's Disease

Alzheimers disease (AD) is the most common cause of dementia. Neuritic amyloid plaques and concomitant chronic inflammation are prominent pathological features of AD. β‐amyloid peptide (Aβ), the major component of plaques, and advanced glycation end products (AGEs), post‐translational protein modifications, are key activators of plaque‐associated inflammation. Aβ, AGEs, S100b, and amphoterin bind to the receptor for AGEs (RAGE), which transmits the signal from RAGE via redox‐sensitive pathways to nuclear factor kappa‐B (NF‐κB)‐regulated cytokines. RAGE‐mediated inflammation caused by glial cells and subsequent changes in neuronal glucose metabolism are likely to be important contributors to neurodegeneration in AD. As long as the neuronal damage is reversible, drugs interfering with the Aβ and AGE–RAGE pathways might be interesting novel therapeutics for the treatment of AD.

Cytotoxicity of Maillard reaction products determined with a peptide spot library

The reaction of reactive carbonyl compounds (RCCs) with lysine and arginine (Maillard reaction) is a common modification of proteins in thermally processed foods. In this study, the toxicity of Maillard reaction products (MRPs) formed from defined amino acids or dipeptides (bound to a cellulose membrane) with ribose, glycerinaldehyde or methylglyoxal was investigated. Murine RAW 264.7 macrophages were cultivated on the cellulose membrane and the effect of MRPs on cell viability was determined. The toxicity of MRPs was dependent on the RCC used and increased in the order of ribose < glycerinaldehyde < methylglyoxal. The dipeptides were more cytotoxic than the amino acids, with Lys-Lys MRPs being the most toxic of all tested MRPs. Cell numbers did not fall below the starting point, indicating that the MRPs rather inhibited proliferation than actually caused cell death. To develop an assay, in which whole membranes with multiple peptide spots could be tested simultaneously, we measured cell numbers on larger cellulose membranes using image analysis of the intracellularly formed formazan crystals. Although this method was technically feasable, it appears that uneven cell attachment on the membrane would require a way to detemine starting cell number by a non-destructive assay to yield more robust data.

Generation of hydrogen peroxide-resistant murine neuroblastoma cells: a target discovery platform for novel neuroprotective genes

Oxidative stress has been suggested to play an important role in the pathogenesis of various neurodegenerative diseases including Alzheimer’s disease (AD). Hydrogen peroxide (H2O2), one of the main reactive oxygen species, is converted into the highly toxic ·OH radical in the presence of redox-active transition metals, which then oxidises nucleic acids, lipids and proteins, leading to neurodegeneration and cell death. There is an urgent need to gain more knowledge about relevant therapeutic targets to combat oxidative stress and it neurotoxic effects, and how this knowledge can be utilized to develop novel neuroprotective therapies for AD. One way to identify new mechanisms combating oxidative stress was via the creation of H2O2-resistant cell lines and identification of the mechanisms responsible for their resistance. However, in most cases catalase overexpression or increased glutathione content was identified as the primary mode of H2O2 resistance in these cell lines. In this study, we have generated six different resistant neuronal cell lines or populations (from the same original murine Neuro2a neuroblastoma line) by exposing cells to increasing concentrations of H2O2 and performing continuous selection for survivors over a period of several months, which appear to have acquired H2O2 resistance based on other, novel mechanisms. These six populations showed a significant, but differential resistance against H2O2 when compared with the parental cell line. Using combinations of catalase-, glutathione synthesis- and glutathione peroxidase-inhibitors it was shown that the increased resistance of Neuro2a-HR cells is not solely based on an increased activity of catalase or the glutathione system, suggesting that their resistance might be based on yet unknown, novel defence mechanisms.

A versatile microglia-neuron co-culture system for the identification of anti-inflammatory neuroprotectants: Application to screening of natural compounds

Introduction: In Alzheimer ́s disease, activated microglia secrete cytokines, including Il-1, Il-6 and TNF as well as reactive oxygen and nitrogen species (ROS/RNS). These factors contribute to alterations in neuronal glucose uptake, inhibition of mitochondrial enzymes, impaired axonal transport, and synaptic signaling. In addition, ROS act as signaling molecules in pro-inflammatory redox-active signal transduction.