Synthetic Thioesters of Thiamine: Promising Tools for Slowing Progression of Neurodegenerative Diseases.

Thiamine (vitamin B1) is essential for the brain. This is attributed to the coenzyme role of thiamine diphosphate (ThDP) in glucose and energy metabolism. The synthetic thiamine prodrug, 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 outcomes in patients with mild Alzheimer's disease. 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, O,S-dibenzoylthiamine (DBT), is even more efficient than 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 metabolites and the clarification of their mechanism of action open extremely promising perspectives in the field of neurodegenerative, neurodevelopmental, and psychiatric conditions. The present review aims to summarize existing data on the neuroprotective effects of thiamine thioesters and give a comprehensive account.

Neuroprotective Effects of Thiamine and Precursors with Higher Bioavailability: Focus on Benfotiamine and Dibenzoylthiamine.

Thiamine (vitamin B1) is essential for brain function because of the coenzyme role of thiamine diphosphate (ThDP) in glucose and energy metabolism. In order to compensate thiamine deficiency, several thiamine precursors with higher bioavailability were developed since the 1950s. 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 antioxidant and anti-inflammatory properties that seem to be mediated by a mechanism independent of the coenzyme function of ThDP. BFT has no adverse effects and improves cognitive outcome in patients with mild Alzheimer's disease (AD). 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. Thiamine thioesters have pleiotropic properties linked to an increase in circulating thiamine concentrations and possibly in hitherto unidentified metabolites in particular 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.

Benfotiamine treatment activates the Nrf2/ARE pathway and is neuroprotective in a transgenic mouse model of tauopathy.

Impaired glucose metabolism, decreased levels of thiamine and its phosphate esters, and reduced activity of thiamine-dependent enzymes, such as pyruvate dehydrogenase, alpha-ketoglutarate dehydrogenase and transketolase occur in Alzheimer's disease (AD). Thiamine deficiency exacerbates amyloid beta (Aß) deposition, tau hyperphosphorylation and oxidative stress. Benfotiamine (BFT) rescued cognitive deficits and reduced Aß burden in amyloid precursor protein (APP)/PS1 mice. In this study, we examined whether BFT confers neuroprotection against tau phosphorylation and the generation of neurofibrillary tangles (NFTs) in the P301S mouse model of tauopathy. Chronic dietary treatment with BFT increased lifespan, improved behavior, reduced glycated tau, decreased NFTs and prevented death of motor neurons. BFT administration significantly ameliorated mitochondrial dysfunction and attenuated oxidative damage and inflammation. We found that BFT and its metabolites (but not thiamine) trigger the expression of Nrf2/antioxidant response element (ARE)-dependent genes in mouse brain as well as in wild-type but not Nrf2-deficient fibroblasts. Active metabolites were more potent in activating the Nrf2 target genes than the parent molecule BFT. Docking studies showed that BFT and its metabolites (but not thiamine) bind to Keap1 with high affinity. These findings demonstrate that BFT activates the Nrf2/ARE pathway and is a promising therapeutic agent for the treatment of diseases with tau pathology, such as AD, frontotemporal dementia and progressive supranuclear palsy.

Enhanced conditioning of adverse memories in the mouse modified swim test is associated with neuroinflammatory changes - Effects that are susceptible to antidepressants.

Deficient learning and memory are well-established pathophysiologic features of depression, however, mechanisms of the enhanced learning of aversive experiences associated with this disorder are poorly understood. Currently, neurobiological mechanisms of enhanced retention of aversive memories during depression, and, in particular, their relation to neuroinflammation are unclear. As the association between major depressive disorder and inflammation has been recognized for some time, we aimed to address whether neuroinflammatory changes are involved in enhanced learning of adversity in a depressive state. To study this question, we used a recently described mouse model of enhanced contextual conditioning of aversive memories, the modified forced swim model (modFST). In this model, the classic two-day forced swim is followed by an additional delayed session on Day 5, where increased floating behaviour and upregulated glycogen synthase kinase-3 (GSK-3) are context-dependent. Here, increased time spent floating on Day 5, a parameter of enhanced learning of the adverse context, was accompanied by hypercorticosteronemia, increased gene expression of GSK-3α, GSK-3ß, c-Fos, cyclooxygenase-1 (COX-1) and pro-inflammatory cytokines interleukin-1 beta (IL-1ß), tumor necrosis factor (TNF), and elevated concentrations of protein carbonyl, a marker of oxidative stress, in the prefrontal cortex and hippocampus. There were significant correlations between cytokine levels and GSK-3ß gene expression. Two-week administration of compounds with antidepressant properties, imipramine (7 mg/kg/day) or thiamine (vitamin B1; 200 mg/kg/day) ameliorated most of the modFST-induced changes. Thus, enhanced learning of adverse memories is associated with pro-inflammatory changes that should be considered for optimizing pharmacotherapy of depression associated with enhanced learning of aversive memories.

Thiamine and benfotiamine prevent stress-induced suppression of hippocampal neurogenesis in mice exposed to predation without affecting brain thiamine diphosphate levels.

Thiamine is essential for normal brain function and its deficiency causes metabolic impairment, specific lesions, oxidative damage and reduced adult hippocampal neurogenesis (AHN). Thiamine precursors with increased bioavailability, especially benfotiamine, exert neuroprotective effects not only for thiamine deficiency (TD), but also in mouse models of neurodegeneration. As it is known that AHN is impaired by stress in rodents, we exposed C57BL6/J mice to predator stress for 5 consecutive nights and studied the proliferation (number of Ki67-positive cells) and survival (number of BrdU-positive cells) of newborn immature neurons in the subgranular zone of the dentate gyrus. In stressed mice, the number of Ki67- and BrdU-positive cells was reduced compared to non-stressed animals. This reduction was prevented when the mice were treated (200mg/kg/day in drinking water for 20days) with thiamine or benfotiamine, that were recently found to prevent stress-induced behavioral changes and glycogen synthase kinase-3ß (GSK-3ß) upregulation in the CNS. Moreover, we show that thiamine and benfotiamine counteract stress-induced bodyweight loss and suppress stress-induced anxiety-like behavior. Both treatments induced a modest increase in the brain content of free thiamine while the level of thiamine diphosphate (ThDP) remained unchanged, suggesting that the beneficial effects observed are not linked to the role of this coenzyme in energy metabolism. Predator stress increased hippocampal protein carbonylation, an indicator of oxidative stress. This effect was antagonized by both thiamine and benfotiamine. Moreover, using cultured mouse neuroblastoma cells, we show that in particular benfotiamine protects against paraquat-induced oxidative stress. We therefore hypothesize that thiamine compounds may act by boosting anti-oxidant cellular defenses, by a mechanism that still remains to be unveiled. Our study demonstrates, for the first time, that thiamine and benfotiamine prevent stress-induced inhibition of hippocampal neurogenesis and accompanying physiological changes. The present data suggest that thiamine precursors with high bioavailability might be useful as a complementary therapy in several neuropsychiatric disorders.

Structural determinants of specificity and catalytic mechanism in mammalian 25-kDa thiamine triphosphatase.

BACKGROUND: Thiamine triphosphate (ThTP) is present in most organisms and might be involved in intracellular signaling. In mammalian cells, the cytosolic ThTP level is controlled by a specific thiamine triphosphatase (ThTPase), belonging to the CYTH superfamily of proteins. CYTH proteins are present in all superkingdoms of life and act on various triphosphorylated substrates. METHODS: Using crystallography, mass spectrometry and mutational analysis, we identified the key structural determinants of the high specificity and catalytic efficiency of mammalian ThTPase. RESULTS: Triphosphate binding requires three conserved arginines while the catalytic mechanism relies on an unusual lysine-tyrosine dyad. By docking of the ThTP molecule in the active site, we found that Trp-53 should interact with the thiazole part of the substrate molecule, thus playing a key role in substrate recognition and specificity. Sea anemone and zebrafish CYTH proteins, which retain the corresponding Trp residue, are also specific ThTPases. Surprisingly, the whole chromosome region containing the ThTPase gene is lost in birds. CONCLUSIONS: The specificity for ThTP is linked to a stacking interaction between the thiazole heterocycle of thiamine and a tryptophan residue. The latter likely plays a key role in the secondary acquisition of ThTPase activity in early metazoan CYTH enzymes, in the lineage leading from cnidarians to mammals. GENERAL SIGNIFICANCE: We show that ThTPase activity is not restricted to mammals as previously thought but is an acquisition of early metazoans. This, and the identification of critically important residues, allows us to draw an evolutionary perspective of the CYTH family of proteins.

Overexpression of CD39 in mouse airways promotes bacteria-induced inflammation.

In airways, the ecto-nucleoside triphosphate diphosphohydrolase CD39 plays a central role in the regulation of physiological mucosal nucleotide concentrations and likely contributes to the control of inflammation because accelerated ATP metabolism occurs in chronic inflammatory lung diseases. We sought to determine whether constant elevated CD39 activity in lung epithelia is sufficient to cause inflammation and whether this affects the response to acute LPS or Pseudomonas aeruginosa exposure. We generated transgenic mice overexpressing human CD39 under the control of the airway-specific Clara cell 10-kDa protein gene promoter. Transgenic mice did not develop any spontaneous lung inflammation. However, intratracheal instillation of LPS resulted in accelerated recruitment of neutrophils to the airways of transgenic mice. Macrophage clearance was delayed, and the amounts of CD8(+) T and B cells were augmented. Increased levels of keratinocyte chemoattractant, IL-6, and RANTES were produced in transgenic lungs. Similarly, higher numbers of neutrophils and macrophages were found in the lungs of transgenic mice infected with P. aeruginosa, which correlated with improved bacteria clearance. The transgenic phenotype was partially and differentially restored by coinstillation of P2X(1) or P2X(7) receptor antagonists or of caffeine with LPS. Thus, a chronic increase of epithelial CD39 expression and activity promotes airway inflammation in response to bacterial challenge by enhancing P1 and P2 receptor activation.

Thiamine triphosphate: a ubiquitous molecule in search of a physiological role.

Thiamine triphosphate (ThTP) was discovered over 60 years ago and it was long thought to be a specifically neuroactive compound. Its presence in most cell types, from bacteria to mammals, would suggest a more general role but this remains undefined. In contrast to thiamine diphosphate (ThDP), ThTP is not a coenzyme. In E. coli cells, ThTP is transiently produced in response to amino acid starvation, while in mammalian cells, it is constitutively produced at a low rate. Though it was long thought that ThTP was synthesized by a ThDP:ATP phosphotransferase, more recent studies indicate that it can be synthesized by two different enzymes: (1) adenylate kinase 1 in the cytosol and (2) FoF1-ATP synthase in brain mitochondria. Both mechanisms are conserved from bacteria to mammals. Thus ThTP synthesis does not seem to require a specific enzyme. In contrast, its hydrolysis is catalyzed, at least in mammalian tissues, by a very specific cytosolic thiamine triphosphatase (ThTPase), controlling the steady-state cellular concentration of ThTP. In some tissues where adenylate kinase activity is high and ThTPase is absent, ThTP accumulates, reaching ≥ 70% of total thiamine, with no obvious physiological consequences. In some animal tissues, ThTP was able to phosphorylate proteins, and activate a high-conductance anion channel in vitro. These observations raise the possibility that ThTP is part of a still uncharacterized cellular signaling pathway. On the other hand, its synthesis by a chemiosmotic mechanism in mitochondria and respiring bacteria might suggest a role in cellular energetics.

A specific inorganic triphosphatase from Nitrosomonas europaea: structure and catalytic mechanism.

The CYTH superfamily of proteins is named after its two founding members, the CyaB adenylyl cyclase from Aeromonas hydrophila and the human 25-kDa thiamine triphosphatase. Because these proteins often form a closed ß-barrel, they are also referred to as triphosphate tunnel metalloenzymes (TTM). Functionally, they are characterized by their ability to bind triphosphorylated substrates and divalent metal ions. These proteins exist in most organisms and catalyze different reactions depending on their origin. Here we investigate structural and catalytic properties of the recombinant TTM protein from Nitrosomonas europaea (NeuTTM), a 19-kDa protein. Crystallographic data show that it crystallizes as a dimer and that, in contrast to other TTM proteins, it has an open ß-barrel structure. We demonstrate that NeuTTM is a highly specific inorganic triphosphatase, hydrolyzing tripolyphosphate (PPP(i)) with high catalytic efficiency in the presence of Mg(2+). These data are supported by native mass spectrometry analysis showing that the enzyme binds PPP(i) (and Mg-PPP(i)) with high affinity (K(d) < 1.5 µm), whereas it has a low affinity for ATP or thiamine triphosphate. In contrast to Aeromonas and Yersinia CyaB proteins, NeuTTM has no adenylyl cyclase activity, but it shares several properties with other enzymes of the CYTH superfamily, e.g. heat stability, alkaline pH optimum, and inhibition by Ca(2+) and Zn(2+) ions. We suggest a catalytic mechanism involving a catalytic dyad formed by Lys-52 and Tyr-28. The present data provide the first characterization of a new type of phosphohydrolase (unrelated to pyrophosphatases or exopolyphosphatases), able to hydrolyze inorganic triphosphate with high specificity.

Reduced Nucleotides, Thiols and O2 in Cellular Redox Balance: A Biochemist's View.

In 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. During catabolic oxidative metabolism, matter-mostly hydrogenated organic molecules-is oxidized 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 reduction to an oxidizing atmosphere, favoring the development of oxidative metabolism in living organisms. We focus on the specific properties of O2 that provide 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, we present the main redox couples involved in cellular redox balance and we discuss the chemical properties underlying their cellular roles and, in particular, 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.

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

BACKGROUND: Thiamine diphosphate (ThDP), an indispensable cofactor for oxidative energy metabolism, is synthesized 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 formation 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 equilibrium 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.

In FUS[1-359]-tg mice O,S-dibenzoyl thiamine reduces muscle atrophy, decreases glycogen synthase kinase 3 beta, and normalizes the metabolome.

Mutations in the gene encoding the RNA/DNA-binding protein Fused in Sarcoma (FUS) have been detected in familial amyotrophic lateral sclerosis (ALS) patients. FUS has been found to be a critical component of the oxidative damage repair complex that might explain its role in neurodegeneration. Here, we examined what impact antioxidant treatment with thiamine (vitamine B1), or its more bioavailable derivative O,S-dibenzoylthiamine (DBT), would have on the hallmarks of pathology in the FUS[1-359]-transgenic mouse model of ALS. From 8-weeks old, in the pre-symptomatic phase of disease, animals received either thiamine, DBT (200 mg/kg/day), or vehicle for 6 weeks. We examined physiological, behavioral, molecular and histological outcomes, as well as the serum metabolome using nuclear magnetic resonance (NMR). The DBT-treated mice displayed improvements in physiological outcomes, motor function and muscle atrophy compared to vehicle, and the treatment normalized levels of brain glycogen synthase kinase-3ß (GSK-3ß), GSK-3ß mRNA and IL-1ß mRNA in the spinal cord. Analysis of the metabolome revealed an increase in the levels of choline and lactate in the vehicle-treated FUS mutants alone, which is also elevated in the cerebrospinal fluid of ALS patients, and reduced glucose and lipoprotein concentrations in the FUS[1-359]-tg mice, which were not the case in the DBT-treated mutants. The administration of thiamine had little impact on the outcome measures, but it did normalize circulating HDL levels. Thus, our study shows that DBT therapy in FUS mutants is more effective than thiamine and highlights how metabolomics may be used to evaluate therapy in this model.

Thiamine triphosphate synthesis in rat brain occurs in mitochondria and is coupled to the respiratory chain.

In animals, thiamine deficiency leads to specific brain lesions, generally attributed to decreased levels of thiamine diphosphate, an essential cofactor in brain energy metabolism. However, another far less abundant derivative, thiamine triphosphate (ThTP), may also have a neuronal function. Here, we show that in the rat brain, ThTP is essentially present and synthesized in mitochondria. In mitochondrial preparations from brain (but not liver), ThTP can be produced from thiamine diphosphate and P(i). This endergonic process is coupled to the oxidation of succinate or NADH through the respiratory chain but cannot be energized by ATP hydrolysis. ThTP synthesis is strongly inhibited by respiratory chain inhibitors, such as myxothiazol and inhibitors of the H(+) channel of F(0)F(1)-ATPase. It is also impaired by disruption of the mitochondria or by depolarization of the inner membrane (by protonophores or valinomycin), indicating that a proton-motive force (Deltap) is required. Collapsing Deltap after ThTP synthesis causes its rapid disappearance, suggesting that both synthesis and hydrolysis are catalyzed by a reversible H(+)-translocating ThTP synthase. The synthesized ThTP can be released from mitochondria in the presence of external P(i). However, ThTP probably does not accumulate in the cytoplasm in vivo, because it is not detected in the cytosolic fraction obtained from a brain homogenate. Our results show for the first time that a high energy triphosphate compound other than ATP can be produced by a chemiosmotic type of mechanism. This might shed a new light on our understanding of the mechanisms of thiamine deficiency-induced brain lesions.

Update on Thiamine Triphosphorylated Derivatives and Metabolizing Enzymatic Complexes.

While the cellular functions of the coenzyme thiamine (vitamin B1) diphosphate (ThDP) are well characterized, the triphosphorylated thiamine derivatives, thiamine triphosphate (ThTP) and adenosine thiamine triphosphate (AThTP), still represent an intriguing mystery. They are present, generally in small amounts, in nearly all organisms, bacteria, fungi, plants, and animals. The synthesis of ThTP seems to require ATP synthase by a mechanism similar to ATP synthesis. In E. coli, ThTP is synthesized during amino acid starvation, while in plants, its synthesis is dependent on photosynthetic processes. In E. coli, ThTP synthesis probably requires oxidation of pyruvate and may play a role at the interface between energy and amino acid metabolism. In animal cells, no mechanism of regulation is known. Cytosolic ThTP levels are controlled by a highly specific cytosolic thiamine triphosphatase (ThTPase), coded by thtpa, and belonging to the ubiquitous family of the triphosphate tunnel metalloenzymes (TTMs). While members of this protein family are found in nearly all living organisms, where they bind organic and inorganic triphosphates, ThTPase activity seems to be restricted to animals. In mammals, THTPA is ubiquitously expressed with probable post-transcriptional regulation. Much less is known about the recently discovered AThTP. In E. coli, AThTP is synthesized by a high molecular weight protein complex from ThDP and ATP or ADP in response to energy stress. A better understanding of these two thiamine derivatives will require the use of transgenic models.

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

BACKGROUND: E. coli 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 approximately 15% of total thiamine. Here, we address the question whether AThTP accumulation in E. coli is triggered by the absence of a carbon source in the medium, the resulting drop in energy charge or other forms of metabolic stress. RESULTS: In minimal M9 medium, E. coli 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 E. coli strain (containing a thermolabile adenylate kinase), the ATP content is very low at 37 degrees 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. CONCLUSIONS: In E. coli, 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.

Role of the Synthetic B1 Vitamin Sulbutiamine on Health.

Sulbutiamine is a thiamine derivative developed in Japan in the mid-60's as a beriberi treatment drug. Since then, different potential applications have been described. For instance, there is some evidence that sulbutiamine can have anti-fatigue, nootropic, and antioxidant effects, which led to its use as a sport supplement (although some authors argue it is actually a masking doping strategy). Moreover, this molecule has been proposed as a possible treatment for some microsporidial infections and even for certain types of cancer. Despite these potential effects, sulbutiamine is still a relatively unknown molecule, which justifies the present review, where we discuss its history and the existing literature on its health applications. We conclude that there is a great potential for sulbutiamine use, well beyond its first described function (to increase thiamine tissue concentration). Indeed, new mechanisms of action have been found, mainly associated with its derivatives. Nevertheless, and although the research on sulbutiamine started 50 years ago, only a limited number of studies were conducted during this time frame. As so, methodological concerns need to be addressed and new studies are necessary, especially randomized controlled trials. Only then will the full potential of this versatile molecule be identified.

Prefrontal cortex inflammation and liver pathologies accompany cognitive and motor deficits following Western diet consumption in non-obese female mice.

AIMS: The high sugar and lipid content of the Western diet (WD) is associated with metabolic dysfunction, non-alcoholic steatohepatitis, and it is an established risk factor for neuropsychiatric disorders. Our previous studies reported negative effects of the WD on rodent emotionality, impulsivity, and sociability in adulthood. Here, we investigated the effect of the WD on motor coordination, novelty recognition, and affective behavior in mice as well as molecular and cellular endpoints in brain and peripheral tissues. MAIN METHODS: Female C57BL/6 J mice were fed the WD for three weeks and were investigated for glucose tolerance, insulin resistance, liver steatosis, and changes in motor coordination, object recognition, and despair behavior in the swim test. Lipids and liver injury markers, including aspartate-transaminase, alanine-transaminase and urea were measured in blood. Serotonin transporter (SERT) expression, the density of Iba1-positive cells and concentration of malondialdehyde were measured in brain. KEY FINDINGS: WD-fed mice exhibited impaired glucose tolerance and insulin resistance, a loss of motor coordination, deficits in novel object exploration and recognition, increased helplessness, dyslipidemia, as well as signs of a non-alcoholic steatohepatitis (NASH)-like syndrome: liver steatosis and increased liver injury markers. Importantly, these changes were accompanied by decreased SERT expression, elevated numbers of microglia cells and malondialdehyde levels in, and restricted to, the prefrontal cortex. SIGNIFICANCE: The WD induces a spectrum of behaviors that are more reminiscent of ADHD and ASD than previously recognized and suggests that, in addition to the impairment of impulsivity and sociability, the consumption of a WD might be expected to exacerbate motor dysfunction that is also known to be associated with adult ADHD and ASD.

Modulation of mitochondrial respiration rate and calcium-induced swelling by new cromakalim analogues.

A cellular model of cardiomyocytes (H9c2 cell line) and mitochondria isolated from mouse liver were used to understand the drug action of BPDZ490 and BPDZ711, two benzopyran analogues of the reference potassium channel opener cromakalim, on mitochondrial respiratory parameters and swelling, by comparing their effects with those of the parent compound cromakalim. For these three compounds, the oxygen consumption rate (OCR) was determined by high-resolution respirometry (HRR) and their impact on adenosine triphosphate (ATP) production and calcium-induced mitochondrial swelling was investigated. Cromakalim did not modify neither the OCR of H9c2 cells and the ATP production nor the Ca-induced swelling. By contrast, the cromakalim analogue BPDZ490 (1) induced a strong increase of OCR, while the other benzopyran analogue BPDZ711 (2) caused a marked slowdown. For both compounds, 1 displayed a biphasic behavior while 2 still showed an inhibitory effect. Both compounds 1 and 2 were also found to decrease the ATP synthesis, with pronounced effect for 2, while cromakalim remained without effect. Overall, these results indicate that cromakalim, as parent molecule, does not induce per se any direct effect on mitochondrial respiratory function neither on whole cells nor on isolated mitochondria whereas both benzopyran analogues 1 and 2 display totally opposite behavior profiles, suggesting that compound 1, by increasing the maximal respiration capacity, might behave as a mild uncoupling agent and compound 2 is taken as an inhibitor of the mitochondrial electron-transfer chain.

Dibenzoylthiamine Has Powerful Antioxidant and Anti-Inflammatory Properties in Cultured Cells and in Mouse Models of Stress and Neurodegeneration.

Thiamine precursors, the most studied being benfotiamine (BFT), have protective effects in mouse models of neurodegenerative diseases. BFT decreased oxidative stress and inflammation, two major characteristics of neurodegenerative diseases, in a neuroblastoma cell line (Neuro2a) and an immortalized brain microglial cell line (BV2). Here, we tested the potential antioxidant and anti-inflammatory effects of the hitherto unexplored derivative O,S-dibenzoylthiamine (DBT) in these two cell lines. We show that DBT protects Neuro2a cells against paraquat (PQ) toxicity by counteracting oxidative stress at low concentrations and increases the synthesis of reduced glutathione and NADPH in a Nrf2-independent manner. In BV2 cells activated by lipopolysaccharides (LPS), DBT significantly decreased inflammation by suppressing translocation of NF-κB to the nucleus. Our results also demonstrate the superiority of DBT over thiamine and other thiamine precursors, including BFT, in all of the in vitro models. Finally, we show that the chronic administration of DBT arrested motor dysfunction in FUS transgenic mice, a model of amyotrophic lateral sclerosis, and it reduced depressive-like behavior in a mouse model of ultrasound-induced stress in which it normalized oxidative stress marker levels in the brain. Together, our data suggest that DBT may have therapeutic potential for brain pathology associated with oxidative stress and inflammation by novel, coenzyme-independent mechanisms.

Benfotiamine and Cognitive Decline in Alzheimer's Disease: Results of a Randomized Placebo-Controlled Phase IIa Clinical Trial.

BACKGROUND: In preclinical models, benfotiamine efficiently ameliorates the clinical and biological pathologies that define Alzheimer's disease (AD) including impaired cognition, amyloid-ß plaques, neurofibrillary tangles, diminished glucose metabolism, oxidative stress, increased advanced glycation end products (AGE), and inflammation. OBJECTIVE: To collect preliminary data on feasibility, safety, and efficacy in individuals with amnestic mild cognitive impairment (aMCI) or mild dementia due to AD in a placebo-controlled trial of benfotiamine. METHODS: A twelve-month treatment with benfotiamine tested whether clinical decline would be delayed in the benfotiamine group compared to the placebo group. The primary clinical outcome was the Alzheimer's Disease Assessment Scale-Cognitive Subscale (ADAS-Cog). Secondary outcomes were the clinical dementia rating (CDR) score and fluorodeoxyglucose (FDG) uptake, measured with brain positron emission tomography (PET). Blood AGE were examined as an exploratory outcome. RESULTS: Participants were treated with benfotiamine (34) or placebo (36). Benfotiamine treatment was safe. The increase in ADAS-Cog was 43% lower in the benfotiamine group than in the placebo group, indicating less cognitive decline, and this effect was nearly statistically significant (p = 0.125). Worsening in CDR was 77% lower (p = 0.034) in the benfotiamine group compared to the placebo group, and this effect was stronger in the APOEɛ4 non-carriers. Benfotiamine significantly reduced increases in AGE (p = 0.044), and this effect was stronger in the APOEɛ4 non-carriers. Exploratory analysis derivation of an FDG PET pattern score showed a treatment effect at one year (p = 0.002). CONCLUSION: Oral benfotiamine is safe and potentially efficacious in improving cognitive outcomes among persons with MCI and mild AD.