In many chronic neurodegenerative diseases including Frontotemporal Dementia and Alzheimer's disease (AD), microglial activation is suggested to be involved in pathogenesis or disease progression. Activated microglia secrete a variety of cytokines, including interleukin-1beta, interleukin-6, and tumor necrosis factor as well as reactive oxygen and nitrogen species (ROS/RNS). ROS and RNS contribute to alterations in neuronal glucose uptake, inhibition of mitochondrial enzymes, a decrease in mitochondrial membrane potential, impaired axonal transport, and synaptic signaling. In addition, ROS act as signaling molecules in pro-inflammatory redox-active signal transduction pathways. To establish a high throughput screening system for anti-inflammatory and neuroprotective compounds, we have constructed an "Enhanced Green Fluorescent protein" (EGFP) expressing neuronal cell line and set up a murine microglia/neuron co-culture system with these EGFP expressing neuronal cells. We show that microglia activation leads to neuronal cell death, which can be conveniently measured by loss of neuronal EGFP fluorescence. Moreover, we used this system to test selected polyphenolic compounds for their ability to downregulate inflammatory markers and to protect neurons against microglial insult. We suggest that this system might allow accelerated drug discovery for the treatment of inflammation-mediated neurodegenerative diseases.
Advanced glycation end products (AGEs) are involved in age-related diseases, including the complications of diabetes and chronic renal impairment with arterial stiffening. Alagebrium chloride (ALT-711) is an AGE-lowering agent with beneficial effects in renal structural and functional parameters in diabetes, decreased diabetes-accelerated atherosclerosis, and age-related myocardial stiffening. ALT-711 exhibits a structural homology to thiamine, and it was suggested to interfere with thiamine metabolism. Thiamine is converted to thiamine diphosphate (TDP) by thiamine diphosphokinase (TDPK). TDP is a cofactor for pyruvate dehydrogenase, α-ketoglutarate dehydrogenase and transketolase. A decreased activity of these enzymes due to TDP deficiency results in disorders such as beriberi and Wernicke-Korsakoff syndrome. Therefore, we investigated whether ALT-711 is an inhibitor of TDPK. Molecular modeling studies showed that ALT-711 fits into the thiamine-binding pocket of TDPK, and there are three interactions between the thiazolium ring and the enzyme, as well as parallel stacking between the phenyl ring and the indole ring of Trp222B. Enzyme kinetic experiments also showed that ALT-711 dose-dependently decreased TDPK activity with K(i)s, calculated by different experiments and fitting models ranging from 0.88 to 1.09 mM. Fitting of the kinetic data favored mixed-mode inhibition with a major role for competitive inhibition. In summary, our results suggest that ALT-711 is a low-affinity inhibitor of TDPK, but is unlikely to interfere with thiamine metabolism at therapeutic concentrations. However, when new AGE-crosslink breakers based on thiamine are designed, care should be taken that they do not act as more potent competitive inhibitors than ALT-711.
Abstract:The enzyme transketolase (sedoheptulose-7-phosphate:D-glyceraldehyde-3-phosphate glycolaldehydetransferase, EC 2.2.1.1) is involved in the pentose phosphate pathway (PPP) and catalyses the transfer of a 2-carbon fragment from a 5-carbon keto sugar (xylulose-5-P) to a 5-carbon aldo sugar (ribose-5-P) to form a 7-carbon keto sugar (sedoheptulose-7-P) and a 3-carbon aldo sugar (glyceraldehyde-3-P). Transketolase requires thiamine pyrophosphate as a co-factor.Advanced glycation endproducts (AGEs) are implicated in the complications of diabetes and aging, primarily via adventitious and crosslinking of tissue proteins. ALT-711 is an AGE crosslink breaker and has been tested as an intervention therapy in established complications of diabetes. It has been noticed that it has a similar structure to that of thiamine and it was hypothesized that it might inhibit transketolase by replacing the active co-factor rendering the enzyme inactive. In this study, we have established a novel microtiter plate format transketolase assay which determines the concentration of NADH by measuring its fluorescence. Using this assay, it was found that ALT-711 does not inhibit the activity of transketolase up to concentration of 5 mM. We conclude that ALT-711 does not interfere with transketolase activity at clinically relevant concentrations.
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