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. Author manuscript; available in PMC: 2022 Aug 28.
Published in final edited form as: Mol Neurobiol. 2017 Nov 11;55(7):5937–5950. doi: 10.1007/s12035-017-0811-0

Impairment of Thiamine Transport at the GUT-BBB-AXIS Contributes to Wernicke’s Encephalopathy

P M Abdul Muneer 1, Saleena Alikunju 2, Heather Schuetz 2, Adam M Szlachetka 2, Xiaotang Ma 1, James Haorah 1
PMCID: PMC9420083  NIHMSID: NIHMS1775673  PMID: 29128903

Abstract

Wernicke’s encephalopathy, a common neurological disease, is caused by thiamine (vitamin B1) deficiency. Neuropathy resulting from thiamine deficiency is a hallmark of Wernicke-Korsakoff syndrome in chronic alcohol users. The underlying mechanisms of this deficiency and progression of neuropathy remain to be understood. To uncover the unknown mechanisms of thiamine deficiency in alcohol abuse, we used chronic alcohol consumption or thiamine deficiency diet ingestion in animal models. Observations from animal models were validated in primary human neuronal culture for neurodegenerative process. We employed radio-labeled bio-distribution of thiamine, qualitative and quantitative analyses of the various biomarkers and neurodegenerative process. In the present studies, we established that disruption of thiamine transport across the intestinal gut blood-brain barrier axis as the cause of thiamine deficiency in the brain for neurodegeneration. We found that reduction in thiamine transport across these interfaces was the cause of reduction in the synthesis of thiamine pyrophosphate (TPP), an active cofactor for pyruvate dehydrogenase E1α (PDHE1α). Our findings revealed that decrease in the levels of PDHE1α cofactors switched on the activation of PD kinase (PDK) in the brain, thereby triggering the neuronal phosphorylation of PDHE1α (p-PDHE1α). Dysfunctional phosphorylated PDHE1α causes the reduction of mitochondrial aerobic respiration that led to neurodegeneration. We concluded that impairment of thiamine transport across the gut-BBB-axis that led to insufficient TPP synthesis was critical to Wernicke-neuropathy, which could be effectively prevented by stabilizing the thiamine transporters.

Keywords: Alcohol, Thiamine deficiency/transporters, Wernicke’s encephalopathy, Pyruvate dehydrogenase, Neurodegeneration, Gut-BBB-axis

Introduction

Alcohol is the most commonly use legalized substance of abuse in the world. The brain is a major target organ of alcohol use since alcohol penetrates across the blood-brain barrier within minutes without any blockade. Heavy chronic alcohol consumption is a well-known cause of the characteristic behavioral, cognitive, and neuropathological complications such as Wernicke-Korsakoff syndrome [1-3], fetal alcohol syndrome [4, 5], and Marchiafava-Bignami disease [6-8]. Marchiafava-Bignami disease is characterized by neuronal degeneration in the corpus callosum of alcohol users. Neuropathy resulting from thiamine (vitamin B1) deficiency is a hallmark of Wernicke-Korsakoff encephalopathy among chronic alcohol users [9, 10]. Wernicke’s encephalopathy in human subjects can also evolve from non-alcohol malnutrition factor besides chronic alcohol use [11, 12]. The common pathological signature in both the cases is manifested by thiamine deficiency and Wernicke-Korsakoff neuropathy. The loss of neurons and white matter microstructure integrity appeared to be prevalent in the frontal cortex or the cerebellum in alcoholics with or without Korsakoff’s syndrome [13, 14]. Studies from various clinical and neuropsychological evaluation concluded that severe anterograde and retrograde amnesia is prevalent in alcoholics Korsakoff syndrome [15]. Such neuropsychological manifestation is believed to be a result of neuropathological outcomes of frontal neuropathy, white matter brain volume shrinkage [16-18] and loss of brain weight in alcoholics [19].

Neurological disease caused by thiamine deficiency is not limited to alcohol-mediated Wernicke-Korsakoff encephalopathy, but it has been implicated in other neurological diseases, such as Alzheimer’s disease (AD) and Parkinson’s disease (PD). As such, thiamine administration has been used as therapy for AD patients [20-23]. Observation of thiamine deficiency in blood cells and diminished levels of thiamine-dependent enzymes in the temporal cortex was implicated as the rationale for thiamine therapy in Alzheimer’s disease patients [24-26]. Similarly, the levels of thiamine in the cerebro-spinal fluid of PD patients were found to be diminished [27]. Thus, long-term parenteral administration of high-dose thiamine appeared to be effective in reversing the motor and non-motor symptoms in PD patients [28, 29].

Thiamine-deficient health complications are not just restricted to Wernicke-Korsakoff syndrome [30, 31], but it also adversely affects multiple tissue organs such as the heart, the muscle, the livers, and the gastrointestinal tract [32-36]. Thus, deleterious effects of thiamine deficiency disease cover multi-facet tissue organ systems because thiamine is an essential nutrient and a critical cofactor for important physiological metabolic pathways of lipids, glucose, and pyruvate metabolism. The adverse effects of thiamine deficiency are well established in cardiovascular, skeletal muscular, and central nervous system diseases, which remains a significant concern for world health problem. Recent studies in the setting of thiamine deficient rodents when challenged with glucose loading in an in vivo imaging seemed to indicate thiamine deficiency-related neurodegeneration, but not neuroinflammation [37]. Other in vitro studies indicated the connection between thiamine deficiency and neuroinflammation via the induction of oxidative stress damage [38-40]. Since alcohol itself is a potent inducer of oxidative stress as well as a mediator of neuroimmune cell inflammation, the role of thiamine deficiency as possible contributing factor is likely to be minimal compare to alcohol as inflammatory agent. Thus, the present studies will focus on the impairment of thiamine transport mechanisms and its deleterious metabolic outcomes in the setting of chronic alcohol consumption.

Interestingly, thiamine deficiency among heavy alcohol users goes up to 80% [41]. In spite of this high incidence, the underlying molecular, cellular, physiological, or tissue organ interplay mechanisms of this deficiency are not well understood. Here, we comprehensively examined the interconnected causative mechanisms of thiamine deficiency in chronic alcohol intake as well as thiamine deficient diet ingestion as positive controls. To rationalize whether alcohol affects the transport of thiamine, we evaluated the changes in bio-distribution of thiamine in all major tissue organs in control, alcohol diet intake, and thiamine deficient diet ingestion. The reduction of thiamine levels in alcohol condition was then validated by impairment of thiamine transports across the intestinal barrier interface and blood-brain barrier (BBB). We then correlated the alcohol-induced diminution of thiamine transports across the BBB to subsequent alterations of enzymatic conversion of thiamine to active thiamine pyrophosphate (TPP) in the brain. Bioavailability of TPP a cofactor of pyruvate dehydrogenase E1α (PDHE1α) appears to act as a switch for pyruvate dehydrogenase kinase (PDK) and pyruvate dehydrogenase phosphatase (PDP). PDK and PDP respectively regulate the phosphorylation (inactive) and dephosphorylation (active) of PDHE1α. We observed that PDK is active during thiamine deficiency, thereby suppressed PDHE1α activity. Our findings suggest that dysfunction of thiamine transporter protein at the barrier interfaces causes the inadequate synthesis of TPP, which is a likely mechanism of Wernicke’s encephalopathy in alcoholics.

Materials and Methods

Reagents

Antibodies to thiamine transporter protein 1 (THTR1) were from Santa Cruz Biotech; THTR2 from Alpha Diagnostic International, San Antonio, TX; thiamine pyrophosphate kinase 1 (TPK1) was purchased from Abgent (San Diego, CA); pyruvate dehydrogenase kinase 1 (PDK1) was from Enzo Life Sciences (Farmingdale, NY); pyruvate dehydrogenase phosphatase (PDP) was from Novus Biologicals (Littleton, CO); antibodies to pyruvate dehydrogenase E1α (PDHE1α), b-actin, von-Willibrand factor (vWF), neurofilament (NF), glucose transporter protein-1 (GLUT1), and NeuN were from Abcam (Cambridge, MA); antibody to phospho-pyruvate dehydrogenase E1a-Ser293 and MAP2 were from Millipore and Invitrogen (Billerica, MA). All secondary Alexa Fluor antibodies were purchased from Invitrogen. Acetyl-L-carnitine (ALC) was purchased from Sigma-Aldrich (St. Louis, MO). 3H-thiamine hydrochloride (5 μCi, 1.85 MBq, Cat. No. ART 0710) was purchased from American Radiolabeled Chemicals, St Louis, MO. Thiamine-deficient diet (TDD) was from Dyets Inc. (Bethlehem, PA).

Cell Culture

Human cortical neurons were obtained from our neural tissue core facility, isolated from elective abortus specimens of human fetal brain tissues. Tissues were obtained in full compliance with the ethical guidelines of the National Institutes of Health (NIH) and University of Nebraska Medical Center. Briefly, dissociated tissues were incubated with 0.25% trypsin for 30 min, neutralized with 10% fetal bovine serum, and further dissociated by trituration. Neurons were cultured on poly-D-lysine pre-coated cover slips and 6-well plates (BD Labware, Bedford, MA) in Neurobasal™ Medium containing 0.5 mM glutamine, 50 μg/ml each of penicillin and streptomycin in combination with GIBCO™B-27 supplements with antioxidants. Purity of neurons was assessed by MAP-2 Abs (Chemicon), which normally showed 100% enrichment of neurons. Neurons were plated on 12-well plates containing glass cover slips (40,000 cells/well) for immunocytochemistry. For protein extractions, neurons were cultured in 6-well plates (0.2 × 106 cells/well). Cell culture media was changed every third day until cells were confluent (10–12 days).

Ethanol Liquid Diets and Thiamine Deficiency Diet Pair-Feeding

Five-week-old male C57BL-6J mice purchased from Jackson Laboratory (Bar Harbor, ME) were maintained in sterile cages under pathogen-free conditions in accordance with the institutional ethical guidelines for care of laboratory animals, National Institutes of health (NIH), and Institutional Animal Care Use Committee. Mice were divided into (1) Lieber–DeCarli control liquid diet, (2) Lieber–DeCarli ethanol (EtOH) liquid diet, (3) thiamine deficient diet (TDD), (4) EtOH + TDD liquid diet, (5) EtOH liquid diet + acetyl-L-carnitine (ALC), and (6) TDD + ALC groups, 15 mice each per group. Mice in ethanol groups were acclimated to Lieber–DeCarli ethanol liquid-diets with increments of 1, 3, and 4% ethanol for a week prior to pair-feeding for 10–12 weeks. Pair feeding was based on daily liquid-diet consumption by ethanol mice. The macronutrient composition of Lieber–DeCarli liquid-diets as percent of total calories consist of 47% carbohydrate, 35% fat, and 18% protein in control, and 35% fat, 28% ethanol caloric intake (4% vol/vol), 19% carbohydrate, and 18% protein in ethanol. Consumption of 0.5–0.7 g of ethanol/kg per day in the present studies was comparable to moderately high alcohol users (0.3–0.4 g of ethanol/kg). A concentration of 1.0 mg of ALC/mL was mixed in the liquid diets.

Bio-Distribution of Thiamine

After 10–12 weeks of pair-feeding, 4 μCi of 3H-thiamine hydrochloride solution in 500 μl saline water was gavaged into each animal consisting of five animals per group without administering anesthesia. Animals were then euthanized after 90–120 min of gavaging with ketamine (100 mg/Kg body weight) and xylazine (10 mg/Kg body weight). Brain tissue, livers, lungs, heart, kidney, and small intestine (jejunum) were surgically removed. All tissue organs were weighed and homogenized in KRPH buffer. A total of 100 μl of KRPH buffer homogenate from each sample was mixed with 4 mL of scintillant fluid for analysis of thiamine uptake in each tissue organ, which was measured in liquid scintillation counter (Beckman). The quantitative data thiamine bio-distribution was then expressed as nCi per gram tissue weight in all tissue organs that were examined in the present studies.

Immunofluorescence and Microscopy

Brain tissue sections (8 μm thickness) containing the external and internal microvessels or human neurons cultured in glass cover slips were washed with PBS and fixed in acetone-methanol (1:1 v/v) fixative for 10 min at 95 °C for immunofluorescence staining. This was followed by incubation with 3% formaldehyde in PBS for 10 min at 25 °C, washed the tissue/cell containing slides, and blocked with 3% bovine serum albumin at 25 °C for 1 h in the presence of 0.1% Triton X-100. Tissues slides were then incubated with primary antibody to goat anti-THTR1, rabbit anti-TPK1, anti-PDHE1α, anti-PDK1, anti-p-PDHE1αSer293, anti-PDH phosphatase, anti-MAP2, anti-GLUT1 (1:250 dilution for all), sheep anti-vWF (1:150 dilution), mouse anti-THTR2, or anti-NF for overnight at 4 °C for probing the respective antigens. After washes with PBS, sample slides were incubated with Alexa Fluor 488 or Alexa Fluor 594 conjugated with anti-mouse or anti-goat or anti-rabbit or anti-sheep immunoglobulin-G (IgG) for 1 h. After washing with PBS sample slides were mounted on immunomount containing DAPI (Invitrogen). Fluorescence microphotographs were captured by fluorescent microscopy (Eclipse TE2000-U, Nikon microscope, Melville, NY) using NIS elements (Nikon, Melville, NY) software.

Western Blotting

Human neuronal cell pellets or cortical brain tissues were lysed with CellLytic-M (Sigma) for 30 min at 4 °C, and centrifuged at 14000×g. The concentrations of protein from cell lysates or brain tissue homogenates were estimated by bicinchoninic acid (BCA) method (Thermo Scientific, Rockford, IL). Protein load was 20 μg/lane in 4–15% SDS-PAGE gradient gels (Thermo Scientific). Molecular size separated proteins were then transferred onto nitrocellulose membranes, blocked with superblock (Thermo Scientific), and incubated overnight with respective primary antibody to THTR1, THTR2, TPK1, PDHE1α, PDK1, p-PDHE1αSer293, PDH phosphatase and b-actin at 4 °C, followed by washes and incubation with horse-radish peroxidase conjugated secondary antibodies for 1 h at 4 °C.

Immunoreactive bands were detected by West Pico chemi-luminescence substrate (Thermo Scientific). Data were quantified as arbitrary densitometry intensity units using the ImageJ software package. All information about the source of antibodies, intended biomarker, catalog numbers, dilutions factors for immunohistochemical staining, and western blotting analyses are listed in Table 1.

Table 1.

Antibodies source, catalog numbers, and dilutions factors for immunohistochemical staining and Western blotting analyses

Antibody Marker for Company Catalog # Dilution for IHC Dilution for WB
vWF Endothelium Abcam ab11713 1:150 1:1000
THTR1&2 Thiamine transporters Alpha diagnostic LS-C148764 1:250 1:1000
TPK1 TPP synthesis Abgent ab2105 1:250 1:1000
PDK1 PDK1 Enzo LifeSci ADI-KAP-PK112-D 1:250 1:1000
PDP PDP Novus Biol NBP1-82912 1:250 1:1000
α-actin α-actin EMD Millipore ABT1487 1:250 1:1000
MAP2 Neuron EMD Millipore MABF216 1:250 1:1000
PDHE1α PDHE1α Abcam ab108362 1:250 1:1000
p-PDHE1α p-PDHE1α EMD Millipore ABS204 1:250 1:1000
Neurofilament neurofilament Abcam ab109390 1:250 1:1000
NeuN neuron nuclei Abcam ab177487 1:250 1:1000
GLUT1 glucose transporter 1 Abcam ab115730 1:250 1:1000
All secondary antibodies Alexa flour Invitrogen 1:5000 1:5000
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Statistical Analysis

GraphPad Prism V5 software (Sorrento Valley, CA) was used for all statistical analysis. Data in graphs are shown as means + SEM; N = X. The numeric values of N are indicated in the respective figure legends. The N values represent the actual number of animals used or experiments performed in cell culture and not the number of replicates per experimental condition. Comparisons between samples were performed by one-way ANOVA with Bonferroni post-hoc tests. Differences between groups with p < 0.05 were considered significant.

Results

Bio-distribution of Thiamine and Effects of Alcohol on Thiamine Uptake

We first established the bio-distribution of thiamine in normal mice by gavaging 4 μCi of 3H-thiamine hydrochloride in saline water. Mice were euthanized at 2.0 h after thiamine administration and harvested all major tissue organs for analysis of thiamine distribution. Thiamine distribution was calculated from total cpms/g tissue divided by total cpms input of 4 μCi of 3H-thiamine. The results were expressed as percent of total cpms input. Thiamine bio-distribution in normal animals was found to be in the order of the intestine (40%), liver (15%), lungs (8%), kidney (12%), heart (5%), brain (2%), and urine (18%) (Fig. 1a). We then examined whether chronic alcohol consumption or thiamine deficiency diet ingestion would interfere thiamine uptake in these major tissue organs. We observed a significant inhibition of thiamine uptake (54–67%) by chronic alcohol intake or by combination of alcohol and thiamine deficient diet (TDD) in all tissue organs compared with those of tissue organs in controls (Fig. 1b-f). Ingestion of TD diet did not further exacerbate the effect of alcohol on thiamine uptake. We observed a higher thiamine uptake in the intestine of TDD ingested animals than those of the controls. The inhibitory effect of alcohol was ameliorated by co-administration of acetyl-L-carnitine (ALC) in most organs, but not intestine. These data suggest the impairment of thiamine transporters at the gut/brain barrier interface by alcohol, but not by TD diet alone.

Fig. 1.

Fig. 1

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Effects of alcohol or thiamine deficiency diet (TDD) on biodistribution of thiamine in different tissue organs: The 3H-thiamine hydrochloride (4 μCi in 500 μl saline water) was gavaged per animal following 10–12 weeks of ethanol/control liquid diet pair-feeding or TDD ingestion. Tissue organ homogenates in KRPH buffer mixed with scintillant fluid were measured for thiamine uptake in liquid scintillation counter, and data were quantified and expressed as nCi/g tissue. a Distribution of thiamine in different tissue organs of normal animal presented in pie diagram. Alterations of thiamine uptake in tissue organs under various experimental conditions, b small intestine, c liver, d blood, e brain, f lungs, g heart, and h kidney. Results are presented as mean values (± SEM, N = 5). Statistical significance indicates *p < 0.05, ***p < 0.001 compared with controls, #p < 0.05, compared with EtOH

Impairment of Thiamine Transporters Causes the Deficiency

The bioavailability of thiamine in the brain neurons relies on the efficiency of transport mechanisms through two biological interface barriers apart from metabolism by livers. They are the gut barrier and the BBB, where the uptake of thiamine is regulated by thiamine transporter protein 1 and 2 (THTR1 and THTR2). To validate that impairment of transporter contributes to thiamine deficiency in Wernicke’s encephalopathy, we evaluated the effect of alcohol on THTR1/THTR2 in small intestine, BBB interface, and neurons. As expected, chronic alcohol intake decreased the immunoreactive staining and protein levels of THTR1 (Fig. 2a-b) and THTR2 (Fig. 2c-d) in the intestine compared with respective controls. Although the significant downregulation of THTR1/THTR2 levels at the BBB endothelium by ethanol was similar to that of the intestine, thiamine transporter THTR1 was more specifically localized at the BBB than the gut barrier (Fig. 3a-d). Thus, inhibition of THTR1 at the BBB and inhibition of THTR2 in the small intestine by alcohol intake seemed to have a negative impact on neuronal thiamine uptake and utilization. This was demonstrated here by qualitative and Western blot analyses in human neuronal culture (Fig. 4a-d). We observed that THTR2 was better expressed in intestine than the BBB endothelium, while THTR1 was more localized at the BBB than in small intestine. Co-localization of glucose transporter 1 (GLUT1), vWF, or MAP2 with THTR1/THTR2 served as respective intestinal epithelial, BBB endothelium, or neuronal marker. ALC prevented the alcohol-induced reduction in THTR1/THTR2 immunoreactivity and THTR2 protein level, but not the THTR1 protein levels in the intestine (Fig. 2a-b). In agreement with thiamine uptake results, we also observed that ingestion of TD diet alone did not affect the levels of THTR1/THTR2.

Fig. 2.

Fig. 2

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Alcohol/TD diets ingestion impairs thiamine transporters in the small intestine. a Representative immunofluorescent staining and microscopy analysis of THTR1 (red) merged with glucose transporter 1 marker, GLUT1 (green) in tissue cross-section of small intestine and b Western blot analysis of THTR1 protein levels in the small intestine tissue homogenates. c Representative immunofluorescent staining of THTR2 (red) merged with glucose transporter 1 marker, GLUT1 (green) in small intestine tissue cross-section and d quantitative validation of THTR2 protein levels in intestine tissue homogenates by Western blot. All quantitative data were normalized to beta-actin and results were expressed as ratio of THTR1/THTR2 to that of beta-actin bands. Results are presented as mean values (± SEM, N = 5). Statistical significance indicates *p < 0.05, ***p < 0.001 compared with controls, $p < 0.05 compared with TDD, and #p < 0.05 compared with EtOH. Scale bar = 50 μm

Fig. 3.

Fig. 3

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Alcohol/TD diets ingestion impairs thiamine transporters at the BBB. a Representative immunofluorescent staining and microscopy analysis of THTR1 (red) merged with GLUT1 (green) in microvessel of brain tissue cross-section and b protein levels of THTR1 in isolated brain microvessels tissue homogenates. c Representative immunofluorescent staining of THTR2 (red) merged with endothelium marker von Willebrand Factor, vWF (green) in microvessel of brain tissue cross-section and d quantitative validation of THTR2 protein levels in isolated brain microvessels tissue homogenates. All quantitative data were normalized to beta-actin and results were expressed as ratio of THTR1/THTR2 to that of beta-actin bands. Results are presented as mean values (± SEM, N = 5). Statistical significance indicates *p < 0.05, ***p < 0.001 compared with controls, $p < 0.05 compared with TDD, and #p < 0.05 compared with EtOH. Scale bar = 50 μm

Fig. 4.

Fig. 4

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Effects of EtOH on neuronal thiamine transporters. a Representative immunofluorescent staining and microscopy analysis of THTR1 (red) merged with neuronal cell marker, MAP2 (green) and DAPI (blue), and b protein levels of THTR1 in neurons. c Representative immunofluorescent staining of THTR2 (red) merged with MAP2 (green) and DAPI (blue), and d quantitative analysis of THTR2 protein levels in neuronal protein extracts. THTR1/THTR2 protein levels were normalized to beta-actin. Results were expressed as ratio of THTR1/THTR2 to that of beta-actin band and are presented as mean values (± SEM, N = 5). Statistical significance indicates *p < 0.05, ***p < 0.001 compared with controls, $p < 0.05 compared with TDD, and #p < 0.05 compared with EtOH. Scale bar = 50 μm

Reduction in Thiamine Impairs Synthesis of Active Thiamine

In order for thiamine to become an active cofactor, thiamine needs to be converted to thiamine pyrophosphate (TPP) by pyrophosphokinase 1 (TPK1; EC 2.7.6.2). TPP and Mg2+ are the critical cofactors for pyruvate dehydrogenase catalytic sites, which play central role for mitochondrial energy generation in living cells. However, the activity of TPK1 is also dependent on the availability of its substrate, thiamine in this case. As such, TPK1 level was expected to decrease during alcohol-induced thiamine deficiency resulting from the impairment of THTR1/THTR2 either in the gut or in the brain. Thus, we examined the changes in TPK1 level in brain tissue section by immunofluorescence staining and microscopy analysis. We also quantified the levels of TPK1 protein in small intestine and brain tissue homogenates from chronic alcohol intake or TD diet ingestion as well as in human primary neuronal culture to complement the in vivo data in a similar experimental setting. In parallel with reduced thiamine levels, we found that TPK1 expression of was decreased in chronic alcohol intake (Fig. 5a). Similarly, alcohol or TD diets ingestion significantly reduced the levels of TPK1 protein in small intestine, brain tissue, and human neurons as confirmed by Western blot analyses (Fig. 5b-d). We also observed that ALC improved the TPK1 expression in alcohol intake, but TPK1 levels were significantly downregulated in TDD due to total unavailability of thiamine.

Fig. 5.

Fig. 5

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EtOH downregulates thiamine pyrophokinase 1 levels (TPK1). a Representative immunofluorescent staining and microscopy analysis of TPK1 (red) and merged DAPI (blue) in brain tissue from EtOH and EtOH + ALC administered mice compared with controls. Western blot analyses of TPK1 protein levels in brain tissue homogenates (b), and human neurons (c). Bar graphs show the results expressed as ratio of TPK1 to that of beta-actin bands, and data are presented as mean SEM (n = 5). Statistically significant, *p < 0.05 compared with controls, and #p < 0.05 compared with EtOH (second bar). Scale bar: 40 μm in all panels

Diminution of TPP Decreases PDHE1α Levels at the Gut and BBB Interface

During the citric acid cycle, PDHE1α catalyzes the conversion of pyruvate to acetyl-coenzyme A when the cofactor thiamine pyrophosphate (TPP) is bound to catalytic site of pyruvate dehydrogenase E1α (PDHE1α). Depletion of TPP resulted from declined thiamine uptake and subsequent TPK1 inactivity that were observed in Figs. 1, 2, and 3 has critical impact on the PDHE1α function. Our results showed that chronic alcohol intake or TD diet ingestion significantly diminished the levels of PDHE1α protein in the gut (Fig. 6a-b) and at the BBB (Fig. 6c-d) compared with controls. The reduction in TPP/TPK1 levels observed in qualitative analyses were in parallel with quantitative data observation in the gut and at the BBB interfaces. Supplementation of ALC somewhat restored PDHE1α levels in ethanol fed animals, but not to the extent of the restoration observed in TDD fed animals. These data suggest that impairment of thiamine transporters by alcohol affects the function of PDHE1α unlike TD diet ingestion, which can be restored by supplementation of ALC or thiamine.

Fig. 6.

Fig. 6

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Alcohol-induced deficiency of thiamine decreased the levels of pyruvate dehydrogenase E1α (PDHE1α) in intestine and brain cortex. a Immunofluorescent staining and microscopy analysis of PDHE1α (red) merged with GLUT1 (green) in intestinal tissue cross-sections. b Western blot analysis of PDHE1α in intestinal tissue homogenates. c Representative immunofluorescent staining and microscopy analysis of PDHE1α in microvessel of brain tissue cross-sections merged with endothelial marker, vWF (green), and d quantitative validation of PDHE1α protein levels in isolated brain microvessel tissue homogenates. All quantitative data were normalized to beta-actin and results were expressed as ratio of PDHE1α to that of beta-actin bands. Results are presented as mean values (± SEM, N = 5). Statistical significance indicates *p < 0.05, **p < 0.001 compared with controls, and #p < 0.05 compared with EtOH. Scale bar = 20 μm

Deficiency of TPP Has Severe Impact in Neuronal Survival

Since TPP is an active cofactor of PDHE1α, thiamine deficiency is directly linked to neuronal PDHE1α function in Wernicke’s neuropathy. As such, we examined the idea that inhibition of thiamine transport at the BBB interface by ethanol would hamper neuronal mitochondrial PDHE1α. The interruption of neuronal mitochondrial energy production is likely the mechanisms of Wernicke’s neuropathy. Our data showed that qualitative expression of PDHE1α protein in neuronal cell body as well as the quantitative levels of PDHE1α in the brain were reduced by ethanol, compared with controls (Fig. 7a-b). As expected, TD diet ingestion diminished PDHE1α level. This is because the synthesis of active cofactor TPP was greatly diminished in the absence of thiamine, and ethanol seemed to exacerbate the effect of TDD on PDHE1α levels. As a proof-of-concept, we validated our in vivo observations with the findings in primary human neuronal culture. Both qualitative (Fig. 8a) and quantitative (Fig. 8b) analyses showed that inhibition of TPP synthesis by ethanol or pyrithiamine significantly reduced the levels of PDHE1α in human neurons, compared with control. The effects of ethanol or TDD on PDHE1α levels were somewhat abrogated by ALC compared with controls. These data suggest that alcohol intake impairs neuronal mitochondrial respiration via TPP-mediated PDHE1α dysfunction, a likely mechanism of Wernicke-neuropathy.

Fig. 7.

Fig. 7

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Alcohol/TDD downregulates PDHE1α protein levels by upregulating the levels of PDHE1α phosphorylation (p-PDHE1α) in the brain. a Representative immunofluorescent staining of PDHE1α (red) in cortical brain tissue merged with neuronal nuclear antigen marker, NeuN (green) and DAPI (blue), and b Western blot analysis of PDHE1α protein levels in mice cortical brain tissue homogenates. c Immunofluorescent staining and microscopy analysis of PDHE1α phosphorylation at amino acid residue Ser293 (p-PDHE1αSer293, red) in cortical brain tissue sections merged with NeuN (green) and DAPI (blue), and d Western blot analysis of p-PDHE1αSer293 levels in cortical brain tissue homogenates. All quantitative data were normalized to beta-actin and results were expressed as ratio of PDHE1α or p-PDHE1αSer293 to those of beta-actin bands. Results are presented as mean values (± SEM, N = 5). Statistically significant, indicates *p < 0.05, **p < 0.001 compared with controls, and #p < 0.05 compared with EtOH. Scale bar = 40 μm

Fig. 8.

Fig. 8

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Alcohol/TDD down-regulates PDHE1α levels by upregulating p-PDHE1α levels in neurons. a Representative immunofluorescent staining and microscopy analysis of PDHE1α (red) in primary human neuronal culture merged with neuronal marker neurofilaments, NF (green) and b changes in PDHE1α protein levels in neuronal lysate proteins in different treatment conditions. c Immunofluorescent staining and microscopy analysis of phosphorylated PDHE1α (p-PDHE1αSer293) (red) in neurons merged with neuronal microtubule marker, MAP-2 (green), and d Western blot analysis of p-PDHE1αSer293 levels in neuronal lysate proteins. All quantitative data were normalized to beta-actin and results were expressed as ratio of PDHE1α or p-PDHE1αSer293 to those of beta-actin bands. Results are presented as mean values (± SEM, N = 5). Statistically significant, indicates *p < 0.05, **p < 0.001 compared with controls, and #p < 0.05 compared with EtOH. Scale bar = 40 μm

Thiamine Deficiency Enhances PDHE1α Phosphorylation.

We next examined the molecular mechanisms of PDHE1α dysfunction in alcohol-induced thiamine deficiency. Pyruvate dehydrogenase (PD) kinase 1 (PDK1) and PD phosphatase (PDP) regulate the deactivation and activation of PDHE1α. We propose that availability of the cofactor TPP acts as a molecular sensor for PDHE1α function, wherein when PDP is active under normal condition PDHE1α is dephosphorylated in its active form. But ethanol-induced TPP deficiency triggers the activation of PDK1 that promotes phosphorylation of PDHE1α (p-PDHE1α) to its inactive form. This is because PDK1 phosphorylates PDHE1-α-subunit at a single Ser-232, – 293, or – 300 residue, which deactivates PDHE1α. Whereas, dephosphorylation of PDHE1-α-subunit at these sites by PDP reactivates PDHE1α. True to our hypothesis, we observed a significant increase of p-PDHE1α protein in neuronal cell body of brain tissue section from ethanol intake animal compared with controls (Fig. 7c). The qualitative increment of p-PDHE1α protein expression elicited by ethanol-induced thiamine deficiency was also confirmed quantitatively in TD diet ingested animal compared with control (Fig. 7d). Similarly, the levels of p-PDHE1α were markedly upregulated in human neuron when exposed to ethanol or inhibition of TPP synthesis by pyrithiamine (Fig. 8c-d). In agreement with the increased levels of PDHE1α phosphorylation, we found that chronic ethanol intake or TD diet ingestion markedly elevated the expression of PDK1 in brain tissue section, brain tissue homogenates, and in human neuronal culture (Fig. 9a-c). In contrast, the levels of PDP in brain tissue homogenates or human neuron were significantly decreased by chronic ethanol intake or TD diet ingestion (Fig. 9a-c). These data indicate that limitation of TPP bio-availability promotes PDK1 activation and PDHE1α phosphorylation. Such dysfunction of PDHE1α hampers neuronal mitochondrial respiration, a likely initiation step for the progression of Wernicke’s neuropathy.

Fig. 9.

Fig. 9

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Increase phosphorylation of PDHE1α is mediated by upregulation of PD kinase-1 (PDK1) and down-regulation of PD phosphatase (PDP) in response to alcohol-induced deficiency of thiamine. a Immunofluorescent staining and microscopy analysis of PDK1 (red) in cortical brain tissue sections from EtOH, EtOH + ALC and controls. Nuclei counter stained with DAPI (blue). Western blot analysis of pyruvate dehydrogenase kinase-1 (PDK1) protein levels in b human neurons and c brain cortical tissue with respective treatment conditions. Alterations of pyruvate dehydrogenase phosphatase (PDP) protein levels in d human neurons and e brain cortical tissue lysates in different experimental conditions. All quantitative data were normalized to beta-actin and results were expressed as ratio of PDK1/PDP to those of beta-actin bands. Results are presented as mean values (± SEM, N = 5). Statistically significant, indicates *p < 0.05, **p < 0.001 compared with controls, and #p < 0.05 compared with EtOH. Scale bar = 40 μm

Discussion

Wernicke-Korsakoff syndrome (WKS) is an irreversible neuropathogenesis caused by thiamine deficiency commonly observed in heavy chronic alcohol users. The causative factors of thiamine deficiency include inadequate intake, inhibition of uptake, and excessive loss due to non-metabolism of thiamine. Supplementation of dietary thiamine is known to be effective for treating the reversible Wernicke’s encephalopathy, but it is not effective for treatment of alcohol-elicited WKS. As such, inadequate thiamine intake alone is insufficient to explain the pathogenesis of WKS progression in alcoholics. The present findings revealed that impairment of thiamine transporters at the gut-BBB interface appeared to be the main cause of thiamine deficiency in alcohol consumption. Limitation of thiamine bio-availability resulted into diminished synthesis of active thiamine pyrophosphate (TPP). Deficiency of TPP then causes the deactivation of PDHE1α in response to PDK activation in the brain, as demonstrated in Fig. 10. Thus, treatment of the irreversible WKS can be achieved more effectively by stabilizing thiamine transporters at the gut-BBB-axis along with thiamine supplement. This is because WKS is not due to inadequate thiamine intake or depletion of thiamine levels in the circulation, but deficiency of thiamine in WKS is due to inefficient transport of thiamine across the BBB and into the brain.

Fig. 10.

Fig. 10

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Proposed mechanisms of alcohol-induced thiamine deficiency triggering the development and progression of Wernicke-Korsakoff Syndrome. Inhibition of THTR1/THTR2 at the gut-BBB-axis decreases the synthesis of PDHE1α active co-factor thiamine pyrophosphate (TPP) in the brain. Deficiency of TPP at PDHE1α catalytic site (see X) triggers PDK1 activation and PDP deactivation, which promotes PDK1-elicited neuronal PDHE1α phosphorylation (inactive PDHE1α). Long-term reduction in brain neuronal oxidative phosphorylation (aerobic respiration) leads to neurodegeneration as observed in Wernicke-neuropathy

Our findings suggest that the gut is critical for active transport of thiamine to other internal organs including the brain. We observed that only about 2% of thiamine is transported across the BBB and into the brain in normal condition. Even this small amount of thiamine transport across the BBB or the gut barrier was significantly inhibited by chronic alcohol intake. This is because thiamine is transported actively under normal physiological condition and not a passive diffusion. This active transport is saturable at as low as 2–4 μM concentration at the gut transport system in normal physiological condition, but when the gut epithelial lining becomes leaky, thiamine can passively diffuse at high concentrations [42,43]. Thus, as little as 2% of thiamine transport into the brain is in line with that of the intestinal thiamine uptake. The active transport of thiamine across the BBB by THTR1 is also saturable at low concentration. As the primary function of thiamine is a cofactor for PDHE1α, the physiological requirement is very minimal but essential, since it is not synthesized in the brain.

Our studies demonstrated that deficiency of thiamine in the brain such as in WKS is initiated by alcohol-elicited impairment of thiamine transporters at these interfaces. Another intriguing observation was that unlike TD diet, the inhibitory effect of chronic alcohol on thiamine transporters (THTR1/THTR2) was not easily reversible at the gut-BBB interfaces. This finding clearly reflects the effective/ineffective treatment of thiamine for reversible Wernicke encephalopathy or irreversible WKS in patients with alcohol use disorders [44]. It has been reported that THTR1, but not THTR2 was prevalent in the intestinal epithelial cells [45]. We found that THTR1 is specific to BBB endothelium thiamine transport system and THTR2 is more specific to intestinal thiamine transport, supporting the findings that intestinal thiamine uptake was impaired in THTR2-deficient mice [42].

It is pertinent to discuss the consequence of brain cellular metabolic event arising from thiamine-derived synthesis of active thiamine pyrophosphate (TPP) by thiamine pyrophosphate kinase 1 (TPK1). A significant reduction in TPK1 levels that we observed in the present studies is suggestive of a deleterious effect of alcohol in TPP synthesis. Ingestion of TD diet also decreased TPK1, an indication of thiamine-dependent TPK1 activation. An impaired synthesis of TPP is expected to diminish mitochondrial Krebs cycle since TPP is a critical cofactor of pyruvate dehydrogenase (PDHE1α). These arguments are substantiated by a significant reduction of PDHE1α protein levels by alcohol/TDD at BBB/in the gut barrier (Fig. 6a-d), in neuronal bodies (Fig. 7a-b), and in human neuronal culture (Fig. 8a-b). These findings suggest that dysfunction of PDHE1α in the brain due to lack of TPP and interruption of energy production is a key factor for Wernicke-neuropathy. Thus, effective coupling of TPP and PDHE1α is critical for catabolic and anabolic cellular energy interplay, such as WKS. In this manner, acetyl-L-carnitine (ALC) appears to protect thiamine transporters at the gut-BBB-axis and stabilizes neuronal PDHE1α function. This is because ALC is a potent stabilizer of mitochondria function, antioxidants, and a neurotransmitter [46-48].

The molecular and cellular mechanisms of WKS or Wernicke’s encephalopathy remain elusive from the perspective of alcohol-elicited dysfunction of PDHE1α in the brain. Reduction of TPP synthesizing enzyme was shown in autopsied brain samples from alcoholic patients with confirmed diagnosis of WKS [49]. Recently, microglial activation resulting from alcohol-induced thiamine deficiency was implicated to inhibition of global glucose metabolism and delirium outcomes in WKS [50, 51]. We propose that under normal condition, coupling of TPP to PDHE1α acts as a molecular sensor for PDP to keep PDHE1α active in its dephosphorylated form. But during alcohol-induced thiamine deficiency, reduction of TPP level switches to PD kinase 1 activation, thereby causes the phosphorylation of PDHE1α (p-PDHE1α) in its inactive form. This argument is supported by our findings that alcohol-induced increase in the levels of p-PDHE1α protein in brain neurons (Fig. 7c-d and Fig. 8a-b) were markedly correlated to increase levels of PDK1 (Fig. 9a-c) and significant decreased levels of PDP (Fig. 9d-e). Phosphorylation of PDHE1α is a post-translational modification, where p-PDHE1α at Ser-232, Ser-293 or Ser-300 sub-unit by PDK inactivates PDHE1α, but dephosphorylation of PDHE1α at all three sites by PDP is required for reactivation.

In conclusion, impairment of thiamine uptake at the gut-BBB interface diminished the synthesis of active TPP. Limitation of TPP bioavailability causes the PDK-elicited phosphorylation of PDHE1α to its inactive form in neurons, and therapeutic application of acetyl-L-carnitine, a mitochondrial energy booster and an antioxidant appeared to reverse the alcohol-elicited deleterious effects. We suggest that stabilizing thiamine transporters at the gut-BBB interface together with thiamine supplement would impact much more effective treatment for Wernicke’s encephalopathy than supplementation of exogenous thiamine alone.

Acknowledgements

The National Institute of Health (NIH/NIAAA) supported the work.

Funding

This work was supported by NIH/NIAAA grant 1R21AA022734-01A1, R21 AA020370-01A1 (to JH).

Footnotes

Ethical Approval and Consent to Participate Elective abortus specimens of human fetal brain tissues were obtained in full compliance with the ethical guidelines of the National Institutes of Health and University of Nebraska Medical Center. No disclosure of the source of abortus tissue or of patient information was possible since only de-identified abortus tissues were obtained from the source. In all instances, informed consent was obtained and maintained by the source.

Availability of Data and Materials All available are presented in this main manuscript.

Competing Interests The authors declare that they have competing interests.

References

  • 1.Kopelman MD (2002) Disorders of memory. Brain: J Neurol 125:2152–2190 [DOI] [PubMed] [Google Scholar]
  • 2.Thomson AD, Marshall EJ (2006) The natural history and patho-physiology of Wernicke’s encephalopathy and Korsakoff’s psychosis. Alcohol Alcohol (Oxford, Oxfordshire) 41:151–158 [DOI] [PubMed] [Google Scholar]
  • 3.Manzo L, Locatelli C, Candura SM, Costa LG (1994) Nutrition and alcohol neurotoxicity. Neurotoxicology 15:555–565 [PubMed] [Google Scholar]
  • 4.Bakhireva LN, Sharkis J, Shrestha S, Miranda-Sohrabji TJ, Williams S, Miranda RC (2017) Prevalence of prenatal alcohol exposure in the state of Texas as assessed by phosphatidylethanol in newborn dried blood spot specimens. Alcohol Clin Exp Res 41:1004–1011 [DOI] [PubMed] [Google Scholar]
  • 5.Bager H, Christensen LP, Husby S, Bjerregaard L (2017) Biomarkers for the detection of prenatal alcohol exposure: a review. Alcohol Clin Exp Res 41:251–261 [DOI] [PubMed] [Google Scholar]
  • 6.Fernandes LMP, Bezerra FR, Monteiro MC, Silva ML, de Oliveira FR, Lima RR, Fontes-Junior EA, Maia CSF (2017) Thiamine deficiency, oxidative metabolic pathways and ethanol-induced neurotoxicity: how poor nutrition contributes to the alcoholic syndrome, as Marchiafava-Bignami disease. Eur J Clin Nutr 71:580–586 [DOI] [PubMed] [Google Scholar]
  • 7.Boloursaz S, Nekooei S, Seilanian Toosi F, Rezaei-Dalouei H, Davachi B, Kazemi S, Abbasi B (2016) Marchiafava-Bignami and alcohol related acute polyneuropathy: the cooccurrence of two rare entities. Case Rep Neurol Med 2016:5848572. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Mehrzad R, Ho MG (2016) Mutism caused by severe demyelination in a patient with Marchiafava-Bignami disease. J Emerg Med 51:e129–e132 [DOI] [PubMed] [Google Scholar]
  • 9.Harper CG, Giles M, Finlay-Jones R (1986) Clinical signs in the Wernicke-Korsakoff complex: a retrospective analysis of 131 cases diagnosed at necropsy. J Neurol Neurosurg Psychiatry 49:341–345 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Sechi G, Serra A (2007) Wernicke’s encephalopathy: new clinical settings and recent advances in diagnosis and management. Lancet Neurol 6:442–455 [DOI] [PubMed] [Google Scholar]
  • 11.Sparacia G, Anastasi A, Speciale C, Agnello F, Banco A (2017) Magnetic resonance imaging in the assessment of brain involvement in alcoholic and nonalcoholic Wernicke’s encephalopathy. World J Radiol 9:72–78 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Nikolakaros G, Ilonen T, Kurki T, Paju J, Papageorgiou SG, Vataja R (2016) Non-alcoholic Korsakoff syndrome in psychiatric patients with a history of undiagnosed Wernicke’s encephalopathy. J Neurol Sci 370:296–302 [DOI] [PubMed] [Google Scholar]
  • 13.Segobin S, Ritz L, Lannuzel C, Boudehent C, Vabret F, Eustache F, Beaunieux H, Pitel AL (2015) Integrity of white matter microstructure in alcoholics with and without Korsakoff's syndrome. Hum Brain Mapp 36:2795–2808 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Baker KG, Harding AJ, Halliday GM, Kril JJ, Harper CG (1999) Neuronal loss in functional zones of the cerebellum of chronic alcoholics with and without Wernicke’s encephalopathy. Neuroscience 91:429–438 [DOI] [PubMed] [Google Scholar]
  • 15.Kopelman MD (2015) What does a comparison of the alcoholic Korsakoff syndrome and thalamic infarction tell us about thalamic amnesia? Neurosci Biobehav Rev 54:46–56 [DOI] [PubMed] [Google Scholar]
  • 16.Logan C, Asadi H, Kok HK, Looby ST, Brennan P, O'Hare A, Thornton J (2016) Neuroimaging of chronic alcohol misuse. J Med Imaging Radiat Oncol 61(4):435–440 [DOI] [PubMed] [Google Scholar]
  • 17.Sutherland GT, Sheedy D, Kril JJ (2014) Neuropathology of alcoholism. Handb Clin Neurol 125:603–615 [DOI] [PubMed] [Google Scholar]
  • 18.Le Berre AP, Pitel AL, Chanraud S, Beaunieux H, Eustache F, Martinot JL, Reynaud M, Martelli C et al. (2015) Sensitive biomarkers of alcoholism’s effect on brain macrostructure: similarities and differences between France and the United States. Front Hum Neurosci 9:354. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Harper CG, Blumbergs PC (1982) Brain weights in alcoholics. J Neurol Neurosurg Psychiatry 45:838–840 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Rommer PS, Fuchs D, Leblhuber F, Schroth R, Greilberger M, Tafeit E, Greilberger J (2016) Lowered levels of carbonyl proteins after vitamin B supplementation in patients with mild cognitive impairment and Alzheimer’s disease. Neurodegener Dis 16:284–289 [DOI] [PubMed] [Google Scholar]
  • 21.Pan X, Chen Z, Fei G, Pan S, Bao W, Ren S, Guan Y, Zhong C (2016) Long-term cognitive improvement after benfotiamine administration in patients with Alzheimer’s disease. Neurosci Bull 32:591–596 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Zhang Q, Yang G, Li W, Fan Z, Sun A, Luo J, Ke ZJ (2011) Thiamine deficiency increases beta-secretase activity and accumulation of beta-amyloid peptides. Neurobiol Aging 32:42–53 [DOI] [PubMed] [Google Scholar]
  • 23.Nolan KA, Black RS, Sheu KF, Langberg J, Blass JP (1991) A trial of thiamine in Alzheimer’s disease. Arch Neurol 48:81–83 [DOI] [PubMed] [Google Scholar]
  • 24.Gold M, Chen MF, Johnson K (1995) Plasma and red blood cell thiamine deficiency in patients with dementia of the Alzheimer’s type. Arch Neurol 52:1081–1086 [DOI] [PubMed] [Google Scholar]
  • 25.Gibson GE, Hirsch JA, Cirio RT, Jordan BD, Fonzetti P, Elder J (2013) Abnormal thiamine-dependent processes in Alzheimer’s disease. Lessons from diabetes. Mol Cell Neurosci 55:17–25 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Butterworth RF, Besnard AM (1990) Thiamine-dependent enzyme changes in temporal cortex of patients with Alzheimer’s disease. Metab Brain Dis 5:179–184 [DOI] [PubMed] [Google Scholar]
  • 27.Jimenez-Jimenez FJ, Molina JA, Hernanz A, Fernandez-Vivancos E, de Bustos F, Barcenilla B, Gomez-Escalonilla C, Zurdo M et al. (1999) Cerebrospinal fluid levels of thiamine in patients with Parkinson’s disease. Neurosci Lett 271:33–36 [DOI] [PubMed] [Google Scholar]
  • 28.Costantini A, Pala MI, Grossi E, Mondonico S, Cardelli LE, Jenner C, Proietti S, Colangeli M et al. (2015) Long-term treatment with high-dose thiamine in Parkinson disease: an open-label pilot study. J Altern Complement Med (New York, NY) 21:740–747 [DOI] [PubMed] [Google Scholar]
  • 29.Costantini A, Pala MI, Compagnoni L, Colangeli M (2013) High-dose thiamine as initial treatment for Parkinson’s disease. BMJ Case Rep 2013. 10.1136/bcr-2013-009289 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Haas RH (1988) Thiamin and the brain. AnnuRevNutr 8:483–515 [DOI] [PubMed] [Google Scholar]
  • 31.Dror V, Rehavi M, Biton IE, Eliash S (2014) Rasagiline prevents neurodegeneration in thiamine deficient rats—a longitudinal MRI study. Brain Res 1557:43–54 [DOI] [PubMed] [Google Scholar]
  • 32.Ahmed M, Azizi-Namini P, Yan AT, Keith M (2015) Thiamin deficiency and heart failure: the current knowledge and gaps in literature. Heart Fail Rev 20:1–11 [DOI] [PubMed] [Google Scholar]
  • 33.DiNicolantonio JJ, Niazi AK, Lavie CJ, O'Keefe JH, Ventura HO (2013) Thiamine supplementation for the treatment of heart failure: a review of the literature. Congest Heart Fail (Greenwich, Conn) 19:214–222 [DOI] [PubMed] [Google Scholar]
  • 34.McCulloch B (2015) High-output heart failure caused by thyrotoxicosis and beriberi. Crit Care Nurs Clin North Am 27:499–510 [DOI] [PubMed] [Google Scholar]
  • 35.Koike H, Watanabe H, Inukai A, Iijima M, Mori K, Hattori N, Sobue G (2006) Myopathy in thiamine deficiency: analysis of a case. J Neurol Sci 249:175–179 [DOI] [PubMed] [Google Scholar]
  • 36.Hernandez-Vazquez AJ, Garcia-Sanchez JA, Moreno-Arriola E, Salvador-Adriano A, Ortega-Cuellar D, Velazquez-Arellano A (2016) Thiamine deprivation produces a liver ATP deficit and metabolic and genomic effects in mice: findings are parallel to those of biotin deficiency and have implications for energy disorders. J Nutrigenet Nutrigenomics 9:287–299 [DOI] [PubMed] [Google Scholar]
  • 37.Zahr NM, Alt C, Mayer D, Rohlfing T, Manning-Bog A, Luong R, Sullivan EV, Pfefferbaum A (2014) Associations between in vivo neuroimaging and postmortem brain cytokine markers in a rodent model of Wernicke's encephalopathy. Exp Neurol 261:109–119 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Liu D, Ke Z, Luo J (2017) Thiamine deficiency and Neurodegeneration: the interplay among oxidative stress, endoplasmic reticulum stress, and autophagy. Mol Neurobiol 54(7):5440–5448 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Wang X, Xu M, Frank JA, Ke ZJ, Luo J (2017) Thiamine deficiency induces endoplasmic reticulum stress and oxidative stress in human neurons derived from induced pluripotent stem cells. Toxicol Appl Pharmacol 320:26–31 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Hazell AS, Faim S, Wertheimer G, Silva VR, Marques CS (2013) The impact of oxidative stress in thiamine deficiency: a multifactorial targeting issue. Neurochem Int 62(5):796–802 [DOI] [PubMed] [Google Scholar]
  • 41.Abdou E, Hazell AS (2015) Thiamine deficiency: an update of pathophysiologic mechanisms and future therapeutic considerations. Neurochem Res 40:353–361 [DOI] [PubMed] [Google Scholar]
  • 42.Reidling JC, Lambrecht N, Kassir M, Said HM (2010) Impaired intestinal vitamin B1 (thiamin) uptake in thiamin transporter-2-deficient mice. Gastroenterology 138:1802–1809 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Hoyumpa AM Jr (1980) Mechanisms of thiamin deficiency in chronic alcoholism. Am J Clin Nutr 33:2750–2761 [DOI] [PubMed] [Google Scholar]
  • 44.Latt N, Dore G (2014) Thiamine in the treatment of Wernicke encephalopathy in patients with alcohol use disorders. Intern Med J 44:911–915 [DOI] [PubMed] [Google Scholar]
  • 45.Subramanya SB, Subramanian VS, Said HM (2010) Chronic alcohol consumption and intestinal thiamin absorption: effects on physiological and molecular parameters of the uptake process. Am J Physiol Gastrointest Liver Physiol 299:G23–G31 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Abdul Muneer PM, Alikunju S, Szlachetka AM, Murrin LC, Haorah J (2011) Impairment of brain endothelial glucose transporter by methamphetamine causes blood-brain barrier dysfunction. Mol Neurodegener 6:23. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Haorah J, Floreani NA, Knipe B, Persidsky Y (2011) Stabilization of superoxide dismutase by acetyl-l-carnitine in human brain endothelium during alcohol exposure: novel protective approach. Free Radic Biol Med 51:1601–1609 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Haorah J, Rump TJ, Xiong H (2013) Reduction of brain mitochondrial beta-oxidation impairs complex I and V in chronic alcohol intake: the underlying mechanism for neurodegeneration. PLoS One 8:e70833. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.Butterworth RF, Kril JJ, Harper CG (1993) Thiamine-dependent enzyme changes in the brains of alcoholics: relationship to the Wernicke-Korsakoff syndrome. Alcohol Clin Exp Res 17:1084–1088 [DOI] [PubMed] [Google Scholar]
  • 50.Qin L, Crews FT (2014) Focal thalamic degeneration from ethanol and thiamine deficiency is associated with neuroimmune gene induction, microglial activation, and lack of monocarboxylic acid transporters. Alcohol Clin Exp Res 38:657–671 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 51.Wijnia JW, Oudman E (2013) Biomarkers of delirium as a clue to diagnosis and pathogenesis of Wernicke-Korsakoff syndrome. Eur J Neurol 20:1531–1538 [DOI] [PubMed] [Google Scholar]

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