Neuroprotective effects of thiamine and precursors with higher bioavailability: focus on benfotiamine and dibenzoylthiamine

Thiamine (vitamin B1) is essential for brain. This is attributed to the coenzyme role of thiamine diphosphate (ThDP) in glucose and energy metabolism. However, thiamine triphosphorylated derivatives thiamine triphosphate and adenosine thiamine triphosphate — the latter discovered in our laboratory — have been described, but heir physiological roles remain unknown. We have recently become interested in thiamine precursors with higher bioavailability. Among these, the thioester benfotiamine (BFT) has been extensively studied and has beneficial effects both in rodent models of neurodegeneration and in human clinical studies. BFT has no known adverse effects and improves cognitive outcome in patients with mild Alzheimer’s disease (AD). The mechanisms of action of BFT remains unknown. Indeed, in cell culture and animal models, BFT has antioxidant and anti-inflammatory properties that seem to be mediated by a mechanism independent of the coenzyme function of ThDP. Recent in vitro studies show that another thiamine thioester, dibenzoylthiamine (DBT) is even more efficient that BFT, especially with respect to its anti-inflammatory potency and is effective at lower concentrations. Thiamine thioesters have pleiotropic properties linked to an increase in circulating thiamine concentrations and possibly in hitherto unidentified open thiazole ring derivatives. The identification of the active neuroprotective derivatives and the clarification of their mechanism of action open extremely promising perspectives in the field of neurodegenerative, neurodevelopmental and psychiatric conditions

Reduced Nucleotides, Thiols and O2 in Cellular Redox Balance: a Biochemist’s View

peer reviewedIn the present review, which is aimed at researchers, teachers and students in life sciences, we try to show how the physicochemical properties of the elements and molecules define the concept of redox balance. Living organism are open systems traversed by fluxes of energy and matter. Dur-ing catabolic oxidative metabolism, matter, mostly hydrogenated organic molecules are oxi-dized and ultimately released as CO2. Electrons are passed over to coupling molecules, such as NAD+ and FAD, whose reduced forms serve as electrons donors in anabolic reactions. Early photosynthetic activity led to the accumulation of O2 and the transformation of the reducing to an oxidizing atmosphere, favoring the development of oxidative metabolism in living organisms. We focus on the specific properties of O2 which provides the chemical energy for the combustion reactions occurring in living cells. We explain the concepts of redox potential and redox balance in complex systems such as living cells and we present the main redox couples involved in cellu-lar redox balance and discuss the chemical properties underlying their cellular roles and, in par-ticular, their antioxidant properties in the defense against reactive oxygen species (ROS). Finally, we try to provide an integrative view emphasizing the interplay between metabolism, oxidative stress and metabolic compartmentation in mammalian cells

Thiamine diphosphate adenylyl transferase from E. coli: functional characterization of the enzyme synthesizing adenosine thiamine triphosphate

BACKGROUND: We have recently identified a new thiamine derivative, adenosine thiamine triphosphate (AThTP), in E. coli. In intact bacteria, this nucleotide is synthesized only in the absence of a metabolizable carbon source and quickly disappears as soon as the cells receive a carbon source such as glucose. Thus, we hypothesized that AThTP may be a signal produced in response to carbon starvation. RESULTS: Here we show that, in bacterial extracts, the biosynthesis of AThTP is carried out from thiamine diphosphate (ThDP) and ADP or ATP by a soluble high molecular mass nucleotidyl transferase. We partially purified this enzyme and characterized some of its functional properties. The enzyme activity had an absolute requirement for divalent metal ions, such as Mn2+ or Mg2+, as well as for a heat-stable soluble activator present in bacterial extracts. The enzyme has a pH optimum of 6.5-7.0 and a high Km for ThDP (5 mM), suggesting that, in vivo, the rate of AThTP synthesis is proportional to the free ThDP concentration. When ADP was used as the variable substrate at a fixed ThDP concentration, a sigmoid curve was obtained, with a Hill coefficient of 2.1 and an S0.5 value of 0.08 mM. The specificity of the AThTP synthesizing enzyme with respect to nucleotide substrate is restricted to ATP/ADP, and only ThDP can serve as the second substrate of the reaction. We tentatively named this enzyme ThDP adenylyl transferase (EC 2.7.7.65). CONCLUSION: This is the first demonstration of an enzyme activity transferring a nucleotidyl group on thiamine diphosphate to produce AThTP. The existence of a mechanism for the enzymatic synthesis of this compound is in agreement with the hypothesis of a non-cofactor role for thiamine derivatives in living cells

Adenylate kinase-independent thiamine triphosphate accumulation under severe energy stress in Escherichia coli

BACKGROUND: Thiamine triphosphate (ThTP) exists in most organisms and might play a role in cellular stress responses. In E. coli, ThTP is accumulated in response to amino acid starvation but the mechanism of its synthesis is still a matter of controversy. It has been suggested that ThTP is synthesized by an ATP-dependent specific thiamine diphosphate kinase. However, it is also known that vertebrate adenylate kinase 1 catalyzes ThTP synthesis at a very low rate and it has been postulated that this enzyme is responsible for ThTP synthesis in vivo. RESULTS: Here we show that bacterial, as vertebrate adenylate kinases are able to catalyze ThTP synthesis, but at a rate more than 106-fold lower than ATP synthesis. This activity is too low to explain the high rate of ThTP accumulation observed in E. coli during amino acid starvation. Moreover, bacteria from the heat-sensitive CV2 strain accumulate high amounts of ThTP (>50% of total thiamine) at 37 degrees C despite complete inactivation of adenylate kinase and a subsequent drop in cellular ATP. CONCLUSION: These results clearly demonstrate that adenylate kinase is not responsible for ThTP synthesis in vivo. Furthermore, they show that E. coli accumulate large amounts of ThTP under severe energy stress when ATP levels are very low, an observation not in favor of an ATP-dependent mechanisms for ThTP synthesis

Adenosine thiamine triphosphate accumulates in Escherichia coli cells in response to specific conditions of metabolic stress

<p>Abstract</p> <p>Background</p> <p><it>E. coli </it>cells are rich in thiamine, most of it in the form of the cofactor thiamine diphosphate (ThDP). Free ThDP is the precursor for two triphosphorylated derivatives, thiamine triphosphate (ThTP) and the newly discovered adenosine thiamine triphosphate (AThTP). While, ThTP accumulation requires oxidation of a carbon source, AThTP slowly accumulates in response to carbon starvation, reaching ~15% of total thiamine. Here, we address the question whether AThTP accumulation in <it>E. coli </it>is triggered by the absence of a carbon source in the medium, the resulting drop in energy charge or other forms of metabolic stress.</p> <p>Results</p> <p>In minimal M9 medium, <it>E. coli </it>cells produce AThTP not only when energy substrates are lacking but also when their metabolization is inhibited. Thus AThTP accumulates in the presence of glucose, when glycolysis is blocked by iodoacetate, or in the presence lactate, when respiration is blocked by cyanide or anoxia. In both cases, ATP synthesis is impaired, but AThTP accumulation does not appear to be a direct consequence of reduced ATP levels. Indeed, in the CV2 <it>E. coli </it>strain (containing a thermolabile adenylate kinase), the ATP content is very low at 37°C, even in the presence of metabolizable substrates (glucose or lactate) and under these conditions, the cells produce ThTP but not AThTP. Furthermore, we show that ThTP inhibits AThTP accumulation. Therefore, we conclude that a low energy charge is not sufficient to trigger AThTP accumulation and the latter can only accumulate under conditions where no ThTP is synthesized. We further show that AThTP production can also be induced by the uncoupler CCCP but, unexpectedly, this requires the presence of pyruvate or a substrate yielding pyruvate (such a D-glucose or L-lactate). Under the conditions described, AThTP production is not different when RelA or SpoT mutants are used.</p> <p>Conclusions</p> <p>In <it>E. coli</it>, AThTP accumulates in response to two different conditions of metabolic stress: lack of energy substrates (or inhibition of their metabolization) and uncoupled pyruvate oxidation. Both conditions prevent bacterial growth. There is no obvious link with the stringent response or catabolite repression.</p

Molecular mechanisms of the non-coenzyme action of thiamin in brain. Biochemical, structural and pathway analysis

Thiamin (vitamin B1) is a pharmacological agent boosting central metabolism through the action of the coenzyme thiamin diphosphate (ThDP). However, positive effects, including improved cognition, of high thiamin doses in neurodegeneration may be observed without increased ThDP or ThDPdependent enzymes in brain. Here, we determine protein partners and metabolic pathways where thiamin acts beyond its coenzyme role. Malate dehydrogenase, glutamate dehydrogenase and pyridoxal kinase were identified as abundant proteins binding to thiamin- or thiazolium-modified sorbents. Kinetic studies, supported by structural analysis, revealed allosteric regulation of these proteins by thiamin and/or its derivatives. Thiamin triphosphate and adenylated thiamin triphosphate activate glutamate dehydrogenase. Thiamin and ThDP regulate malate dehydrogenase isoforms and pyridoxal kinase. Thiamin regulation of enzymes related to malate-aspartate shuttle may impact on malate/citrate exchange, responsible for exporting acetyl residues from mitochondria. Indeed, bioinformatic analyses found an association between thiamin- and thiazolium-binding proteins and the term acetylation. Our interdisciplinary study shows that thiamin is not only a coenzyme for acetyl-CoA production, but also an allosteric regulator of acetyl-CoA metabolism including regulatory acetylation of proteins and acetylcholine biosynthesis. Moreover, thiamin action in neurodegeneration may also involve neurodegeneration-related 14-3-3, DJ-1 and β-amyloid precursor proteins identified among the thiamin- and/or thiazolium-binding proteins

Simultaneous versus solitary pharmacological manipulation of NMDA- and AMPA- receptors: effects of new drugs on contextual learning and its extinction

Both the attenuation of the NMDA-receptor mediated transmission via low affinity blockade mechanism, and the stimulation of AMPA receptor-mediated signaling were shown to result in beneficial neurobiological effects, such as an enhancement of memory and neurogenesis. We aimed to compare the effects of acute pharmacological manipulations of these mechanisms, exerted simultaneously or solely in mice, on learning of two mouse tasks with distinct predominant dependency on either glutamate receptor subtype. In a step-down avoidance task, memantine, low affinity NMDA receptor blocker (5 mg/kg), but not ampakine QQX (5 mg/kg) increased memory scores. In contrast, extinction of contextual fear conditioning was significantly enhanced by the latter, but not by the first drug. Among four new isothiourea derivates used at the doses 0.5-1 mg/kg, one compound that showed a maximal potency with respect to both glutamatergic mechanisms, as well as dimebon (1 mg/kg), had the most prominent memory enhancing effects. Thus, simultaneous low affinity blocade of the NMDA receptor and stimulation of AMPA-mediated transmission can result in eminent pro-cognitive activities. These data point to the importance of multi-target drug mechanism in the regulation of cognitive functions and suggest its potential for clinical implications

Product inhibition of mammalian thiamine pyrophosphokinase is an important mechanism for maintaining thiamine diphosphate homeostasis

peer reviewedBackground: Thiamine diphosphate (ThDP), an indispensable cofactor for oxidative energy metabolism, is syn- thesized through the reaction thiamine + ATP ⇆ ThDP + AMP, catalyzed by thiamine pyrophosphokinase 1 (TPK1), a cytosolic dimeric enzyme. It was claimed that the equilibrium of the reaction is in favor of the for- mation of thiamine and ATP, at odds with thermodynamic calculations. Here we show that this discrepancy is due to feedback inhibition by the product ThDP. Methods: We used a purified recombinant mouse TPK1 to study reaction kinetics in the forward (physiological) and for the first time also in the reverse direction. Results: Keq values reported previously are strongly underestimated, due to the fact the reaction in the forward direction rapidly slows down and reaches a pseudo-equilibrium as ThDP accumulates. We found that ThDP is a potent non-competitive inhibitor (Ki ≈ 0.4 μM) of the forward reaction. In the reverse direction, a true equi- librium is reached with a Keq of about 2 × 10− 5, strongly in favor of ThDP formation. In the reverse direction, we found a very low Km for ThDP (0.05 μM), in agreement with a tight binding of ThDP to the enzyme. General significance: Inhibition of TPK1 by ThDP explains why intracellular ThDP levels remain low after administration of even very high doses of thiamine. Understanding the consequences of this feedback inhibition is essential for developing reliable methods for measuring TPK activity in tissue extracts and for optimizing the therapeutic use of thiamine and its prodrugs with higher bioavailability under pathological conditions

Functionally Fractal Urban Networks: Geospatial Co-location and Homogeneity of Infrastructure

Just as natural river networks are known to be globally self-similar, recent research has shown that human-built urban networks, such as road networks, are also functionally self-similar, and have fractal topology with power-law node-degree distributions (p(k) = a k). Here we show, for the first time, that other urban infrastructure networks (sanitary and storm-water sewers), which sustain flows of critical services for urban citizens, also show scale-free functional topologies. For roads and drainage networks, we compared functional topological metrics, derived from high-resolution data (70,000 nodes) for a large US city providing services to about 900,000 citizens over an area of about 1,000 km2. For the whole city and for different sized subnets, we also examined these networks in terms of geospatial co-location (roads and sewers). Our analyses reveal functional topological homogeneity among all the subnets within the city, in spite of differences in several urban attributes. The functional topologies of all subnets of both infrastructure types resemble power-law distributions, with tails becoming increasingly power-law as the subnet area increases. Our findings hold implications for assessing the vulnerability of these critical infrastructure networks to cascading shocks based on spatial interdependency, and for improved design and maintenance of urban infrastructure networks