ERK1/2 modulation in the central nervous system of male and female thiamine-deficient mice with amprolium.

Thiamine deficiency experimental models focus on using the pyrithiamine analog in male rodents, making the thiamine deficiency effects in females and the use of other thiamine antagonists, such as amprolium, unknown. We investigated the impact of thiamine deficiency with amprolium in the cerebral cortex and thalamus of male and female mice by evaluating the modulation of ERK1/2 phosphorylation. The animals were exposed for 20 days to thiamine-deficient chow with different doses of amprolium (20, 40, 60 and 80 mg/kg) and at different treatment periods (five, 10, 15 or 20 days) at a dose of 60 mg/kg. After treatments, ERK1/2 phosphorylation was analyzed by western blot. In male mice, we observed a progressive increase in ERK1/2 phosphorylation in both the cerebral cortex and thalamus in response to the dose of amprolium. In females, ERK1/2 phosphorylation did not progressively increase in response to the amprolium dosage. However, an increase in phosphorylation at the higher doses of 60 and 80 mg/kg was observed. We observed a more intense increase in ERK1/2 phosphorylation in males' cerebral cortex and thalamus from 10 days onwards. In females, the ERK1/2 modulation profiles were similar. The results show that thiamine deficiency induction with amprolium is efficient, compatible with other recognized models that use pyrithiamine, showing changes in cell signaling in the nervous system. The study showed differences in response to thiamine deficiency with amprolium between male and female mice in relation to ERK1/2 phosphorylation and demonstrated that females respond positively to the method and can also be used as model animals.

Identification of a novel pyrithiamine resistance marker gene thiI for genome co-editing in Aspergillus oryzae.

Marker genes are essential for gene modification and genome editing of microorganisms. In Aspergillus oryzae, a widely used host for enzyme production, only a few marker genes can be used for positive selection. One of these genes, the pyrithiamine (PT) resistance marker gene thiA, is not useful for CRISPR/Cas9 genome editing because of its unique resistance-conferring mechanism. In this study, a novel PT resistance marker was investigated considering its potential applications in genome editing. A mutant resistant to PT was selected from UV-mutagenized A. oryzae RIB40. Whole genome analysis was conducted on the mutants, and a novel candidate gene for PT resistance was identified. This candidate gene exhibited similarity to the thiamine transporter gene thi9 of Schizosaccharomyces pombe and was designated as thiI. A thiI loss-of-function mutant was generated using the CRISPR/Cas9 genome editing system to investigate its effect on PT resistance. This mutant showed PT resistance and exhibited no growth defect or auxotrophy. The thiI gene was further investigated for its use as a selection marker in genome co-editing. Ribonucleoprotein complex comprising recombinant Cas9 nuclease and sgRNA targeting thiI or another target gene (wA or sreA) was prepared and simultaneously introduced into A. oryzae RIB40. thiI and target gene double loss-of-function mutants were efficiently selected on PT-containing medium. thiI was shown to be a useful marker gene in A. oryzae for use in genome editing. This study is expected to provide insights, which will promote basic research and industrial applications of A. oryzae.

Thiamin transport in Helicobacter pylori lacking the de novo synthesis of thiamin.

Helicobacter pylori lacks the genes involved in the de novo synthesis of thiamin, and is therefore a thiamin auxotroph. The PnuT transporter, a member of the Pnu transporter family, mediates the uptake of thiamin across the membrane. In the genome of H. pylori, the pnuT gene is clustered with the thiamin pyrophosphokinase gene thi80. In this study, we found that [3H]thiamin is incorporated into the H. pylori SS1 strain via facilitated diffusion with a Km value of 28 µM. The incorporation of radioactive thiamin was inhibited to some extent by 2-methyl-4-amino-5-hydroxymethylpyrimidine or pyrithiamine, but was largely unaffected by thiamin phosphate or thiamin pyrophosphate. RT-PCR analysis demonstrated that the pnuT and thi80 genes are cotranscribed as a single transcript. The estimated Km value for thiamin in the thiamin pyrophosphokinase activity exerted by the recombinant Thi80 protein was 0.40 µM, which is much lower than the Km value of thiamin transport in H. pylori cells. These findings suggested that the incorporated thiamin from the environment is efficiently trapped by pyrophosphorylation to make the transport directional. In addition, the thiamin transport activity in the pnuT-deficient H. pylori strain was less than 20 % of that in the wild-type strain at extracellular thiamin concentration of 1 µM, but the incorporated scintillation signals of the pnuT-deficient strain with 100 nM [3H]thiamin were nearly at the background level. We also found that the pnuT-deficient strain required 100-times more thiamin to achieve growth equal to that of the wild-type. These findings reflect the presence of multiple routes for entry of thiamin into H. pylori, and PnuT is likely responsible for the high-affinity thiamin transport and serves as a target for antimicrobial agents against H. pylori.

A Dual Selection Marker Transformation System Using Agrobacterium tumefaciens for the Industrial Aspergillus oryzae 3.042.

Currently, the genetic modification of Aspergillus oryzae is mainly dependent on protoplastmediated transformation (PMT). In this study, we established a dual selection marker system in an industrial A. oryzae 3.042 strain by using Agrobacterium tumefaciens-mediated transformation (ATMT). We first constructed a uridine/uracil auxotrophic A. oryzae 3.042 strain and a pyrithiamine (PT)-resistance binary vector. Then, we established the ATMT system by using uridine/uracil auxotrophy and PT-resistance genes as selection markers. Finally, a dual selection marker ATMT system was developed. This study demonstrates a useful dual selection marker transformation system for genetic manipulations of A. oryzae 3.042.

Vitamin B1-deficient mice show impairment of hippocampus-dependent memory formation and loss of hippocampal neurons and dendritic spines: potential microendophenotypes of Wernicke-Korsakoff syndrome.

Patients with severe Wernicke-Korsakoff syndrome (WKS) associated with vitamin B1 (thiamine) deficiency (TD) show enduring impairment of memory formation. The mechanisms of memory impairment induced by TD remain unknown. Here, we show that hippocampal degeneration is a potential microendophenotype (an endophenotype of brain disease at the cellular and synaptic levels) of WKS in pyrithiamine-induced thiamine deficiency (PTD) mice, a rodent model of WKS. PTD mice show deficits in the hippocampus-dependent memory formation, although they show normal hippocampus-independent memory. Similarly with WKS, impairments in memory formation did not recover even at 6 months after treatment with PTD. Importantly, PTD mice exhibit a decrease in neurons in the CA1, CA3, and dentate gyrus (DG) regions of the hippocampus and reduced density of wide dendritic spines in the DG. Our findings suggest that TD induces hippocampal degeneration, including the loss of neurons and spines, thereby leading to enduring impairment of hippocampus-dependent memory formation.

Identification of vitamin B1 metabolism as a tumor-specific radiosensitizing pathway using a high-throughput colony formation screen.

Colony formation is the gold standard assay for determining reproductive cell death after radiation treatment, since effects on proliferation often do not reflect survival. We have developed a high-throughput radiosensitivity screening method based on clonogenicity and screened a siRNA library against kinases. Thiamine pyrophosphokinase-1 (TPK1), a key component of Vitamin B1/thiamine metabolism, was identified as a target for radiosensitization. TPK1 knockdown caused significant radiosensitization in cancer but not normal tissue cell lines. Other means of blocking this pathway, knockdown of thiamine transporter-1 (THTR1) or treatment with the thiamine analogue pyrithiamine hydrobromide (PyrH) caused significant tumor specific radiosensitization. There was persistent DNA damage in cells irradiated after TPK1 and THTR1 knockdown or PyrH treatment. Thus this screen allowed the identification of thiamine metabolism as a novel radiosensitization target that affects DNA repair. Short-term modulation of thiamine metabolism could be a clinically exploitable strategy to achieve tumor specific radiosensitization.

Reconstitution of a secondary metabolite biosynthetic pathway in a heterologous fungal host.

Expression of multiple genes involved in a particular metabolic pathway in a heterologous host facilitates the study of fungal secondary metabolite biosynthesis and production of useful compounds. Two plasmids with different selection markers, argB and the pyrithiamine resistance marker, are used to transform Aspergillus oryzae allowing for expression of up to three genes simultaneously.

Development and refinement of a high-efficiency gene-targeting system for Aspergillus flavus.

An efficient gene-targeting system based on impairment of the nonhomologous end-joining pathway and the orotidine monophosphate decarboxylase gene (pyrG) in Aspergillus flavus was established. It was achieved by replacing the ku70 gene with the Aspergillus oryzae pyrithiamine resistance (ptr) gene and by inserting the Aspergillus parasiticus cypA gene into the pyrG locus. The utility of this system was demonstrated by disruption of nine candidate genes for conidial pigment biosynthesis. The gene-targeting frequencies ranged from 80 to 100%. Two linked genes on chromosome 4, wA and olgA, were confirmed to be involved in pigment formation. In contrast to the parental strain which produced yellowish-green conidia, the knockout mutants produced white and olive-green conidia, respectively. The system was further refined by restoring the pyrithiamine sensitivity and uracil auxotrophy in the A. flavus transformation recipient with an engineered pyrG marker. The improvement allowed gene manipulation using the reusable pyrG marker as shown by the restoration of laeA-mediated aflatoxin production in an A. flavus laeA-deleted mutant.

Comparative characteristic of thiamine antagonists on apoptosis induction in different types of nerve cell lines.

Abnormalities in oxidative metabolism and inflammation accompany many neurodegenerative diseases. The mechanisms of neurodegeneration induced by thiamine deficiency remain incompletely elucidated. The susceptibility of various types of nerve cells to thiamine (vitamin B) antagonists--oxythiamine (OT), pyrithiamine (PT) and amprolium (Am) was investigated. Four cell lines (neuronally differentiated rat PC-12, rat astrocytes DITNC, neuronally differentiated human SH-SY5Y and human astrocytic cells 1321N1) were used for experiments as neural cell models. When different cell types were cultivated with thiamine antagonists, a significant decrease of viability was detected in a time- and dose-dependent manner as demonstrated by the WST-1 colorimetric assay. These data were similar to those of caspase 3 activity and DNA fragmentation induced by thiamine antagonists. All tested cell lines were more vulnerable to OT and PT than to Am. Am displayed a pronounced damaging action on neuronal cells and had a modest influence on astrocytes. The last observation gives the basis to suppose, that neuronal cells need external arrival of thiamine more than astrocytes. Thus, the results testify that various types of nerve cells have different susceptibility to the thiamine antagonists and this relates to extent of apoptosis development.

Thiamine deficiency caused by thiamine antagonists triggers upregulation of apoptosis inducing factor gene expression and leads to caspase 3-mediated apoptosis in neuronally differentiated rat PC-12 cells.

Recent evidence suggests that alterations in oxidative metabolism induced by thiamine deficiency lead to neuronal cell death. However, the molecular mechanisms underlying this process are still under extensive investigation. Here, we report that rat pheochromocytoma PC-12 cells differentiated in the presence of NGF into neurons undergo apoptosis due to thiamine deficiency caused by antagonists of thiamine - amprolium, pyrithiamine and oxythiamine. Confocal laser scanning fluorescence microscopy revealed that annexin V binds to PC-12 cells in presence of thiamine antagonists after 72 h incubation. Results also show that thiamine antagonists trigger upregulation of gene expression of mitochondrial-derived apoptosis inducing factor, DNA fragmentation, cleavage of caspase 3 and translocation of active product to the nucleus. We therefore propose that apoptosis induced by amprolium, pyrithiamine or oxythiamine occurs via the mitochondria-dependent caspase 3-mediated signaling pathway. In addition, our data indicate that pyrithiamine and oxythiamine are more potent inducers of apoptosis than amprolium.

Interaction of thiamine deficiency and voluntary alcohol consumption disrupts rat corpus callosum ultrastructure.

The relative roles of alcohol and thiamine deficiency in causing brain damage remain controversial in alcoholics without the Wernicke-Korsakoff syndrome. Experimental control over alcohol consumption and diet are impossible in humans but can be accomplished in animal models. This experiment was designed to differentiate the separate and combined effects on the macro- and ultrastructure of the corpus callosum of thiamine deficiency and voluntary alcohol consumption. Adult male alcohol-preferring (P) rats (9 chronically alcohol-exposed and 9 water controls) received a thiamine-deficient diet for 2 weeks. There were four groups: five rats previously exposed to alcohol were treated with pyrithiamine (a thiamine phosphorylation inhibitor); five rats never exposed to alcohol were treated with pyrithiamine; four alcohol-exposed rats were treated with thiamine; and four rats never exposed to alcohol were treated with thiamine. On day 14, thiamine was restored in all 18 rats; 2 weeks later the 10 pyrithiamine-treated rats received intraperitoneal thiamine. The rats were perfused 61 days post-pyrithiamine treatment at age 598 days. Brains were dissected and weight and volumes were calculated. Sagittal sections were stained to measure white matter structures. The corpus callosum was examined using transmission electron microscopy to determine density of myelinated fibers, fiber diameter, and myelin thickness. The corpus callosum in the alcohol/pyrithiamine group was significantly thinner, had greater fiber density, higher percentage of small fibers, and myelin thinning than in the alcohol/thiamine and water/thiamine groups. Several measures showed a graded effect, where the alcohol/pyrithiamine group had greater pathology than the water/pyrithiamine group, which had greater pathology than the two thiamine-replete groups. Across all 16 rats, thinner myelin sheaths correlated with higher percentage of small fibers. Myelin thickness and axon diameter together accounted for 71% of the variance associated with percentage of small fibers. Significant abnormalities in the alcohol/pyrithiamine group and lack of abnormality in the alcohol-exposed/thiamine-replete group indicate that thiamine deficiency caused white matter damage. The graded abnormalities across the dually to singly treated animals support a compounding effect of alcohol exposure and thiamine depletion, and indicate the potential for interaction between alcohol and thiamine deficiency in human alcohol-related brain damage.

Structure of the eukaryotic thiamine pyrophosphate riboswitch with its regulatory ligand.

Riboswitches are untranslated regions of messenger RNA, which adopt alternate structures depending on the binding of specific metabolites. Such conformational switching regulates the expression of proteins involved in the biosynthesis of riboswitch substrates. Here, we present the 2.9 angstrom-resolution crystal structure of the eukaryotic Arabidopsis thaliana thiamine pyrophosphate (TPP)-specific riboswitch in complex with its natural ligand. The riboswitch specifically recognizes the TPP via conserved residues located within two highly distorted parallel "sensor" helices. The structure provides the basis for understanding the reorganization of the riboswitch fold upon TPP binding and explains the mechanism of resistance to the antibiotic pyrithiamine.

Thiamine pyrophosphate riboswitches are targets for the antimicrobial compound pyrithiamine.

Thiamine metabolism genes are regulated in numerous bacteria by a riboswitch class that binds the coenzyme thiamine pyrophosphate (TPP). We demonstrate that the antimicrobial action of the thiamine analog pyrithiamine (PT) is mediated by interaction with TPP riboswitches in bacteria and fungi. For example, pyrithiamine pyrophosphate (PTPP) binds the TPP riboswitch controlling the tenA operon in Bacillus subtilis. Expression of a TPP riboswitch-regulated reporter gene is reduced in transgenic B. subtilis or Escherichia coli when grown in the presence of thiamine or PT, while mutant riboswitches in these organisms are unresponsive to these ligands. Bacteria selected for PT resistance bear specific mutations that disrupt ligand binding to TPP riboswitches and derepress certain TPP metabolic genes. Our findings demonstrate that riboswitches can serve as antimicrobial drug targets and expand our understanding of thiamine metabolism in bacteria.

Thiamine deficiency in the pathogenesis of chronic ethanol-associated cerebellar damage in vitro.

Nutritional deficiencies associated with long-term ethanol consumption may cause neuronal damage in ethanol-dependent individuals. Thiamine deficiency, in particular, is thought to contribute to ethanol-associated cerebellar degeneration, although damage may occur in adequately nourished alcoholics. Thus, the present study examined the effects of thiamine depletion and ethanol exposure on cytotoxicity in rat cerebellum. Organotypic cerebellar slice cultures were treated starting at 25 days in vitro with 100 mM ethanol for 11 days or 10 days followed by a 24-h withdrawal period. This exposure paradigm has previously been shown in hippocampal slice cultures to result in spontaneous cytotoxicity upon ethanol withdrawal. Additional cerebellar cultures were exposed to the thiamine depleting agent pyrithiamine (10-500 microM) for 10 or 11 days, some in the presence of ethanol exposure or withdrawal. Other cultures were co-exposed to thiamine (1-100 microM), 500 microM pyrithiamine, and ethanol for 10 or 11 days. The results demonstrated that neither 11-day ethanol treatment nor withdrawal from 10-day exposure significantly increased cerebellar cytotoxicity, as measured by propidium iodide fluorescence. The 11-day treatment with 100 or 500 microM pyrithiamine significantly increased propidium iodide fluorescence approximately 21% above levels observed in control tissue. Cultures treated with both ethanol (11 days or 10 days plus withdrawal) and 500 microM pyrithiamine displayed a marked increase in cytotoxicity approximately 60-90% above levels observed in control cultures. Pyrithiamine and ethanol-induced cytotoxicity was prevented in cultures co-exposed to thiamine (10-100 microM) for the duration of pyrithiamine treatment. Findings from this report suggest that the cerebellum may be more sensitive to the toxic effects of thiamine deficiency, as compared with alcohol withdrawal, associated with alcohol dependence.

Thiamine-regulated gene expression of Aspergillus oryzae thiA requires splicing of the intron containing a riboswitch-like domain in the 5'-UTR.

Exogenous thiamine regulates Aspergillus oryzae thiA, which is involved in thiamine synthesis. One of the two introns in its 5'-untranslated region (5'-UTR) contains motifs (regions A and B) highly conserved among fungal thiamine biosynthesis genes. Deletion of either region relieved the repression by thiamine and thiamine inhibited intron splicing, suggesting that regions A and B are required for efficient splicing. Furthermore, transcript splicing was essential for thiA gene expression. These observations suggest a novel gene expression regulatory mechanism in filamentous fungi, in which exogenous thiamine controls intron splicing to regulate gene expression. Interestingly, regions A and B constitute a part of a thiamine pyrophosphate-binding riboswitch-like domain that has been quite recently found in the 5'-UTR of thiA.

Neuropathological study of the role of mast cells and histamine-positive neurons in selective vulnerability of the thalamus and inferior colliculus in thiamine-deficient encephalopathy.

The purpose of the present study was to examine the role of histamine in the pathogenesis of experimental thiamine-deficient encephalopathy. By studying sagittal serial sections the authors were able to examine the topographical relationship between histamine-positive neurons and fibers, the number of mast cells, and localized lesions in the thalamus (TH) and inferior colliculus (IC). Adult rats were given a thiamine-deficient diet and pyrithiamine was given intraperitoneally (30 microg/100 g bodyweight per day), and the distribution of vulnerable regions and petechial bleeding was histologically examined by reconstruction of the sagittal serial sections. The distribution of mast cells and histamine-positive neurons and fibers was examined immunohistochemically in control rats, and compared between the vulnerable and non-vulnerable regions of the TH and tectum. Changes in the aforementioned measures during the thiamine-deficient state were also examined. The blood-brain barrier was examined using antibodies against rat endothelial barrier antigen (EBA) and albumin. The density of histamine-positive fibers in the vulnerable regions of the TH and IC was very low and not different from the non-vulnerable regions, and the number of mast cells was significantly higher in the lateral portion of the TH than the medial portion of the TH. The numbers of mast cells increased on days 7-10 after the start of the experiment, and significantly decreased on days 14-21. Histamine-positive neurons and fibers in the TH and IC also had the same changes. Bleeding of the IC occurred exclusively around arteries, and perivenous bleeding was absent. Albumin exudation and suppression of EBA expression of capillaries were found in the spongy lesions of the TH and IC. The role of histamine in selective vulnerability of the TH and IC in experimental thiamine-deficient encephalopathy was not supported. Findings in the present study suggest that the spongy change is a primary event, and vascular changes are secondary.

Three thiamine analogues differently alter thiamine transport and metabolism in nervous tissue: an in vivo kinetic study using rats.

Thiamine (T) analogues pyrithiamine, oxythiamine or amprolium in amounts 10-1000 times higher than labelled T, were i.p. injected into rats together with 14C-T (30 microg; 46.25 KBq). The radioactivity associated with T and its phosphoesters in the plasma and cerebral cortex, brainstem, cerebellum, and sciatic nerve were determined at time intervals from 0.25 to 240 h from injection. The experimental data obtained were processed with a mathematical compartmental model that calculated the fractional rate constants. These are the amount of content in a given compartment that is replaced in 1 h and expressed in per hour. The results showed that all three analogues inhibited thiamine entry from plasma. Instead, oxythiamine enhanced T phosphorylation to T pyrophosphate (TPP); amprolium and oxythiamine enhanced TPP dephosphorylation to monophosphate (TMP); pyrithiamine reduced TPP dephosphorylation and TMP formation, while none of the analogues modified TMP dephosphorylation to T. In conclusion, in living rats, the action of T analogues was much more complex than could be expected from their structure and action in vitro.

Physiological, biochemical and morphological studies of Baltic salmon yolk-sac fry with an experimental thiamine deficiency: relations to the M74 syndrome.

Sea-run Baltic salmon (Salmo salar) populations are suffering from the M74 syndrome, a reproduction disorder affecting both broodfish and their progeny. The syndrome is usually manifested during the middle part of the yolk-sac fry stage and has been shown to be associated with a thiamine (vitamin B(1)) deficiency. Development of the disease is reversible by thiamine treatments of broodfish or progeny. This study aimed at investigating the ability of the thiamine antagonist pyrithiamine, administered by microinjections 3 days after hatch, to cause M74-like signs i.e. typical clinical symptoms, high mortality rates and histopathological changes. Furthermore, the effects of pyrithiamine on hepatic activities of the thiamine-dependent enzyme transketolase (TK), the glucose-6-phosphate dehydrogenase (G6PDH) and the cytochrome P4501A (CYP1A) were evaluated. Six family groups with differing thiamine status were sampled on three occasions during the yolk-sac fry stage. All pyrithiamine exposed groups, with the exception of the one with the highest thiamine concentration, showed M74-like symptoms and suffered from high mortality. Enzyme activities were not different in pyrithiamine groups as compared with controls. However, the TK-activities were strongly associated with the thiamine concentrations. The G6PDH-activity demonstrated small variations with the highest activities in the M74-groups. The [TK]/[G6PDH]-ratios were considerably lower in the M74-groups than in the healthy controls, indicating an imbalance between the oxidative and the non-oxidative part of the pentose-phosphate shunt due to a deficit in thiamine. The pyrithiamine-injections induced several M74-like symptoms including incoordination, lethargy, whitened liver and yolk-sac precipitates. They also caused high mortality rates, in addition to lowered glycogen levels and increased prevalence of necrotic brain cells. Moreover, the study demonstrates that the TK, G6PDH and CYP1A-activities are associated with the thiamine content.

Transformation of Aspergillus sp. and Trichoderma reesei using the pyrithiamine resistance gene (ptrA) of Aspergillus oryzae.

A pyrithiamine (PT) resistance gene (ptrA) was cloned from a PT resistant mutant of Aspergillus oryzae and was useful as a dominant selectable marker for transformation of all A. oryzae wild type strain as well as A. nidulans. For further study, we examined whether or not ptrA could be used as the transformation marker in other species of filamentous fungi. Two types of plasmid, which contain ptrA as a selectable marker, were constructed, and the transformation experiments were done with them. One is an integrative plasmid, pPTRI, and another is the autonomously replicating plasmid pPTRII, which contains AMA1. PT-resistant transformants were obtained in the cases of A. kawachii, A. terreus, A. fumigatus, and Trichoderma reesei as hosts with pPTRI and pPTRII. Furthermore, a beta-glucuronidase (GUS) gene was introduced into A. kawachii and A. fumigatus using pPTRII. Almost all the transformants turned blue on GUS assay plates. These results indicate that ptrA can also be used for some other filamentous fungi besides A. oryzae and A. nidulans.

Selective down-regulation of the astrocyte glutamate transporters GLT-1 and GLAST within the medial thalamus in experimental Wernicke's encephalopathy.

Although earlier studies on thiamine deficiency have reported increases in extracellular glutamate concentration in the thalamus, a vulnerable region of the brain in this disorder, the mechanism by which this occurs has remained unresolved. Treatment with pyrithiamine, a central thiamine antagonist, resulted in a 71 and 55% decrease in protein levels of the astrocyte glutamate transporters GLT-1 and GLAST, respectively, by immunoblotting in the medial thalamus of day 14 symptomatic rats at loss of righting reflexes. These changes occurred prior to the onset of convulsions and pannecrosis. Loss of both GLT-1 and GLAST transporter sites was also confirmed in this region of the thalamus at the symptomatic stage using immunohistochemical methods. In contrast, no change in either transporter protein was detected in the non-vulnerable frontal parietal cortex. These effects are selective; protein levels of the astrocyte GABA transporter GAT-3 were unaffected in the medial thalamus. In addition, astrocyte-specific glial fibrillary acidic protein (GFAP) content was unchanged in this brain region, suggesting that astrocytes are spared in this disorder. Loss of GLT-1 or GLAST protein was not observed on day 12 of treatment, indicating that down-regulation of these transporters occurs within 48 h prior to loss of righting reflexes. Finally, GLT-1 content was positively correlated with levels of the neurofilament protein alpha-internexin, suggesting that early neuronal drop-out may contribute to the down-regulation of this glutamate transporter and subsequent pannecrosis. A selective, focal loss of GLT-1 and GLAST transporter proteins provides a rational explanation for the increase in interstitial glutamate levels, and may play a major role in the selective vulnerability of thalamic structures to thiamine deficiency-induced cell death.