Protocatechuic Acid Prevents Some of the Memory-Related Behavioural and Neurotransmitter Changes in a Pyrithiamine-Induced Thiamine Deficiency Model of Wernicke-Korsakoff Syndrome in Rats.

The purpose of this research was to investigate the effects of protocatechuic acid (PCA) at doses of 50 and 100 mg/kg on the development of unfavourable changes in cognitive processes in a pyrithiamine-induced thiamine deficiency (PTD) model of the Wernicke-Korsakoff syndrome (WKS) in rats. The effects of PCA were assessed at the behavioural and biochemical levels. Behavioural analysis was conducted using the Foot Fault test (FF), Bar test, Open Field test, Novel Object Recognition test (NOR), Hole-Board test and Morris Water Maze test (MWM). Biochemical analysis consisting of determination of concentration and turnover of neurotransmitters in selected structures of the rat CNS was carried out using high-performance liquid chromatography. PTD caused catalepsy (Bar test) and significantly impaired motor functions, leading to increased ladder crossing time and multiplied errors due to foot misplacement (FF). Rats with experimentally induced WKS showed impaired consolidation and recall of spatial reference memory in the MWM test, while episodic memory related to object recognition in the NOR was unimpaired. Compared to the control group, rats with WKS showed reduced serotonin levels in the prefrontal cortex and changes in dopamine and/or norepinephrine metabolites in the prefrontal cortex, medulla oblongata and spinal cord. PTD was also found to affect alanine, serine, glutamate, and threonine levels in certain areas of the rat brain. PCA alleviated PTD-induced cataleptic symptoms in rats, also improving their performance in the Foot Fault test. In the MWM, PCA at 50 and 100 mg/kg b.w. improved memory consolidation and the ability to retrieve acquired information in rats, thereby preventing unfavourable changes caused by PTD. PCA at both tested doses was also shown to have a beneficial effect on normalising PTD-disrupted alanine and glutamate concentrations in the medulla oblongata. These findings demonstrate that certain cognitive deficits in spatial memory and abnormalities in neurotransmitter levels persist in rats that have experienced an acute episode of PTD, despite restoration of thiamine supply and long-term recovery. PCA supplementation largely had a preventive effect on the development of these deficits, to some extent also normalising neurotransmitter concentrations in the brain.

Stage-dependent alterations of progenitor cell proliferation and neurogenesis in an animal model of Wernicke-Korsakoff syndrome.

Alcohol-induced Wernicke-Korsakoff syndrome (WKS) culminates in bilateral diencephalic lesion and severe amnesia. Using the pyrithiamine-induced thiamine deficiency (PTD) animal paradigm of WKS, our laboratory has demonstrated hippocampal dysfunction in the absence of gross anatomical pathology. Extensive literature has revealed reduced hippocampal neurogenesis following a neuropathological insult, which might contribute to hippocampus-based learning and memory impairments. Thus, the current investigation was conducted to determine whether PTD treatment altered hippocampal neurogenesis in a stage-dependent fashion. Male Sprague-Dawley rats were assigned to one of 4 stages of thiamine deficiency based on behavioral symptoms: pre-symptomatic stage, ataxic stage, early post-opisthotonus stage, or the late post-opisthotonus stage. The S-phase mitotic marker 5'-bromo-2'-deoxyuridine (BrdU) was administered at the conclusion of each stage following thiamine restoration and subjects were perfused 24 hours or 28 days after BrdU to assess cellular proliferation or neurogenesis and survival, respectively. Dorsal hippocampal sections were immunostained for BrdU (proliferating cell marker), NeuN (neurons), GFAP (astrocytes), Iba-1 (microglia), and O4 (oligodendrocytes). The PTD treatment increased progenitor cell proliferation and survival during the early post-opisthotonus stage. However, levels of neurogenesis were reduced during this stage as well as the late post-opisthotonus stage where there was also an increase in astrocytogenesis. The diminished numbers of newly generated neurons (BrdU/NeuN co-localization) was paralleled by increased BrdU cells that did not co-localize with any of the phenotypic markers during these later stages. These data demonstrate that long-term alterations in neurogenesis and gliogenesis might contribute to the observed hippocampal dysfunction in the PTD model and human WKS.

Thiamine deficiency increases ß-secretase activity and accumulation of ß-amyloid peptides.

Thiamine pyrophosphate (TPP) and the activities of thiamine-dependent enzymes are reduced in Alzheimer's disease (AD) patients. In this study, we analyzed the relationship between thiamine deficiency (TD) and amyloid precursor protein (APP) processing in both cellular and animal models of TD. In SH-SY5Y neuroblastoma cells overexpressing APP, TD promoted maturation of ß-site APP cleaving enzyme 1 (BACE1) and increased ß-secretase activity which resulted in elevated levels of ß-amyloid (Aß) as well as ß-secretase cleaved C-terminal fragment (ß-CTF). An inhibitor of ß-secretase efficiently reduced TD-induced up-regulation of Aß and ß-CTF. Importantly, thiamine supplementation reversed the TD-induced alterations. Furthermore, TD treatment caused a significant accumulation of reactive oxygen species (ROS); antioxidants suppressed ROS production and maturation of BACE1, as well as TD-induced Aß accumulation. On the other hand, exogenous Aß(1-40) enhanced TD-induced production of ROS. A study on mice indicated that TD also caused Aß accumulation in the brain, which was reversed by thiamine supplementation. Taken together, our study suggests that TD could enhance Aß generation by promoting ß-secretase activity, and the accumulation of Aß subsequently exacerbated TD-induced oxidative stress.

Acetyl-CoA and acetylcholine metabolism in nerve terminal compartment of thiamine deficient rat brain.

The decrease of pyruvate and ketoglutarate dehydrogenase complex activities is the main cause of energy and acetyl-CoA deficits in thiamine deficiency-evoked cholinergic encephalopathies. However, disturbances in pathways of acetyl-CoA metabolism leading to appearance of cholinergic deficits remain unknown. Therefore, the aim of this work was to investigate alterations in concentration and distribution of acetyl-CoA and in acetylcholine metabolism in brain nerve terminals, caused by thiamine deficits. They were induced by the pyrithiamine, a potent inhibitor of thiamine pyrophosphokinase. The thiamine deficit reduced metabolic fluxes through pyruvate and ketoglutarate dehydrogenase steps, yielding deficits of acetyl-CoA in mitochondrial and cytoplasmic compartments of K-depolarized nerve terminals. It also inhibited indirect transport of acetyl-CoA though ATP-citrate lyase pathway being without effect on its direct Ca-dependent transport to synaptoplasm. Resulting suppression of synaptoplasmic acetyl-CoA correlated with inhibition of quantal acetylcholine release (r = 0.91, p = 0.012). On the other hand, thiamine deficiency activated non-quantal acetylcholine release that was independent of shifts in intraterminal distribution of acetyl-CoA. Choline acetyltransferase activity was not changed by these conditions. These data indicate that divergent alterations in the release of non-quantal and quantal acetylcholine pools from thiamine deficient nerve terminals could be caused by the inhibition of acetyl-CoA and citrate synthesis in their mitochondria. They in turn, caused inhibition of acetyl-CoA transport to the synaptoplasmic compartment through ATP-citrate lyase pathway yielding deficits of cholinergic functions.

Thiamine deficiency results in downregulation of the GLAST glutamate transporter in cultured astrocytes.

Pyrithiamine-induced thiamine deficiency (TD) is a well-established model of Wernicke's encephalopathy in which a glutamate-mediated excitotoxic mechanism may play an important role in determining selective vulnerability. In order to examine this possibility, cultured astrocytes were exposed to TD and effects on glutamate transport and metabolic function were studied. TD led to decreases in cellular levels of thiamine and thiamine diphosphate (TDP) after 24 h of treatment and decreased activities of the TDP-dependent enzymes alpha-ketoglutarate dehydrogenase and transketolase after 4 and 7 days, respectively. TD treatment for 10 days led to a reversible decrease in the uptake of [(3)H]-D-aspartate, a nonmetabolizable analogue of glutamate. Kinetic analysis revealed that the uptake inhibition was caused by a 47% decrease in the V(max) for uptake of [(3)H]-D-aspartate, with no change in the K(m) value. Immunoblotting showed that this decrease in uptake was due to an 81% downregulation of the astrocyte-specific GLAST glutamate transporter. Loss of uptake activity and GLAST protein were blocked by treatment with the protein kinase C inhibitor H7, while exposure to DCG IV, a group II metabotropic glutamate receptor (mGluR) agonist, resulted in improvement of [(3)H]-D-aspartate uptake and a partial reversal of transporter downregulation. These results are consistent with our recent in vivo findings of a loss of astrocytic glutamate transporters in TD and provide evidence that TD conditions may increase phosphorylation of GLAST, contributing to its downregulation. In addition, manipulation of group II mGluR activity may provide an important strategy in the treatment of this disorder.

[Effect of pyrithiamine on rat sciatic nerve. (I) Morphological changes during the early stage of thiamine deficiency (author's transl)].

We observed under light and electron microscopes morphological changes in the rat sciatic nerve during the early stages of a thiamine deficient state as induced by a pyrithiamine (PT: 50 microgram/100g X 6 days) and thiamine deficient diet (TDD). We simultaneously determined thiamine levels in the whole sciatic nerve of rats. Experiments were undertaken with normal control, TDD (rats fed a TDD), PT (PT treated rats) and PTD (PT treated rats fed a TDD) groups. Microscopically, there were numerous shrunken myelinated axons with myelin ovoids of folds in the PT group and many swollen ones in the PTD group. Electron microscopically, we found more advanced lesions in the PTD group than in the other groups. These ultrastructural changes were swelling of Schwann cells, enlarged rough-surfaced endoplasmic reticulum, axonal degeneration with shrinkage, loss of organelle or abnormal myelin sheath, and proliferation of fibroblasts. The thiamine level in the PTD group decreased to 18 approximately 30% that of control in proportion to the morphological changes. On the other hand, the thiamine level in the PT group (55 approximately 61%) decreased slightly more than that of the TDD group (50 approximately 56%), but changes in morphology were vice versa. These results suggest that the morphological changes in the sciatic nerve caused by PT-induced thiamine deficiency differs from changes seen in cases of dying-back neuropathy caused by TDD-induced deficiency, and that PT itself directly affects the nervous system

[Effect of pyrithiamine on rat sciatic nerve. (II) Morphological changes during the last stages of thiamine deficiency (author's transl)].

We observed under light and electron microscopes morphological changes in the rat sciatic nerve during the last stages of a thiamine deficient state as induced by pyrithiamine (PT: 50 microgram/100g X 11 days) and thiamine deficient diet (TDD), in which rats experienced severe tetanic convulsions. Experiments were undertaken with normal control, PT (PT treated rats) and PTD (PT treated rats fed a TDD) groups. Grossly, 4 out of 9 rats in the PTD group had severe tetanic convulsions on the 12th day and there were no neurologic signs in the PT group. Microscopically, many of the myelinated axons in the animals in the PT group showed shrinkage with myelin ovoids and folds, but in the PTD group, there was swelling as well as shrinkage. Despite the difference in general-symptoms between the PT and the PTD groups, axonal degeneration in the both groups, as determined electron microscopically was almost to the same degree and the convulsed rats in the PTD group showed the severest changes in the myelin sheath. These ultrastructural changes included swelling or vacuolation of Schwann cells, axonal degeneration with the appearance of a myelin like structure, collection of neuro-tubules or vacuoles, invasion of Schwann cell or/and myelin fragments into the periaxonal space, and active phagocytosis by the macrophages. These results suggest that PT directly affects the nervous system and that the dysfunction of the sciatic nerve following PT-induced thiamine deficiency originates from the central nervous system.