Dietary restriction attenuates the neuronal loss, induction of heme oxygenase-1 and blood–brain barrier breakdown induced by impaired oxidative metabolism

Experimental thiamine deficiency (TD) is a model of impaired oxidative metabolism associated with region-selective neuronal loss in the brain. Oxidative stress is a prominent feature of TD neuropathology, as evidenced by the accumulation of heme oxygenase-1 (HO-1), ferritin, reactive iron and superoxide dismutase in microglia, nitrotyrosine and 4-hydroxynonenal in neurons, as well as induction of endothelial nitric oxide synthase within the vulnerable areas. Dietary restriction (DR) reduces oxidative stress in several organ systems including the brain. DR increases lifespan and reduces neurodegeneration in a variety of models of neuronal injury. The possibility that DR can protect vulnerable neurons against TD-induced oxidative insults has not been tested. The current studies tested whether approximately 3 months of DR (60% of ad libitum intake) altered the response to TD. Six month-old ad libitum-fed or dietary restricted C57BL/6 mice received a thiamine-deficient diet either ad libitum, or under a DR regimen respectively for eleven days. The TD mice also received daily injections of the thiamine antagonist pyrithiamine. Control ad libitum-fed or DR mice received an unlimited amount, or 60% of ad libitum intake, respectively, of thiamine-supplemented diet. As in past studies, TD produced region-selective neuronal loss (−60%), HO-1 induction, and IgG extravasation in the thalamus of ad libitum-fed mice. DR attenuated the TD-induced neuronal loss (−30%), HO-1 induction and IgG extravasation in the thalamus. These studies suggest that oxidative damage is critical to the pathogenesis of TD, and that DR modulates the extent of free radical damage in the brain. Thus, TD is an important model for studying the relationship between aging, oxidative stress and nutrition.     

Reversal of thiamine deficiency-induced neurodegeneration

Neurodegenerative diseases are characterized by abnormalities in oxidative processes, region-selective neuron loss, and diminished thiamine-dependent enzymes. Thiamine deficiency (TD) diminishes thiamine dependent enzymes, alters mitochondrial function, impairs oxidative metabolism, and causes selective neuronal death. In mice, the time course of TD-induced changes in neurons and microglia were determined in the brain region most sensitive to TD. Significant neuron loss (29%) occurred after 8 or 9 days of TD (TD8-9) and increased to 90% neuron loss by TD10-11. The number of microglia increased 16% by TD8 and by nearly 400% on TD11. Hemeoxygenase-1 (HO-1)-positive microglia were not detectable at TD8, yet increased dramatically coincident with neuron loss. To test the duration of TD critical for irrevocable changes, mice received thiamine after various durations of TD. Thiamine administration on TD8 blocked further neuronal loss and induction of HO-1-positive microglia, whereas other microglial changes persisted. Thiamine only partially reversed effects on TD9, and was ineffective on TD10-11. These studies indicate that irreversible steps leading to neuronal death and induction of HO-1-positive microglia occur on TD9. The results indicate that TD induces alterations in neurons. endothelial cells, and microglia contemporaneously. This model provides a unique paradigm for elucidating the molecular mechanisms involved in neuronal commitment to neuronal death cascades and contributory microglial activity.     

Vascular factors are critical in selective neuronal loss in an animal model of impaired oxidative metabolism

Thiamine deficiency (TD) models the cellular and molecular mechanisms by which chronic oxidative deficits lead to death of select neurons in brain. Region- and cell-specific oxidative stress and vascular changes accompany the TD-induced neurodegeneration. The current studies analyzed the role of oxidative stress in initiating these events by testing the role of intercellular adhesion molecule-1 (ICAM-1) and endothelial nitric oxide synthase (eNOS) in the selective neuronal loss that begins in the submedial thalamic nucleus of mice. Oxidative stress to microvessels is known to induce eNOS and ICAM-1. TD increased ICAM-1 immunoreactivity in microvessels within the submedial nucleus and adjacent regions 1 day prior to the onset of neuronal loss. On subsequent days, the pattern of ICAM-1 induction overlapped that of neuronal loss, and of induction of the oxidative stress marker heme oxygenase-1 (HO-1). The intensity and extent of ICAM-1 and HO-1 induction progressively spread in parallel with the neuronal death in the thalamus. Targeted disruption of ICAM-1 or eNOS gene, but not the neuronal NOS gene, attenuated the TD-induced neurodegeneration and HO-1 induction. TD induced ICAM-1 in eNOS knockout mice, but did not induce eNOS in mice lacking ICAM-1. These results demonstrate that in TD, an ICAM-1-dependent pathway of eNOS induction leads to oxidative stress-mediated death of metabolically compromised neurons. Thus, TD provides a useful model to help elucidate the role of ICAM-1 and eNOS in the selective neuronal death in diseases in which oxidative stress is implicated.     

Oxidative stress is associated with region-specific neuronal death during thiamine deficiency.

Thiamine deficiency (TD) is a model of chronic impairment of oxidative metabolism and selective neuronal loss. TD leads to region-specific neuronal death and elevation of inducible nitric oxide synthase (iNOS) in macrophages/microglia in mouse brain. Identification of the initial site of neuronal death in the submedial thalamic nucleus allowed us to test the role of iNOS and oxidative stress in TD-induced neuronal death. The pattern of neuronal loss, which begins after 9 days of TD, overlapped with induction of the oxidative stress marker heme oxygenase-1 (HO-1) in microglia. Neuronal death and microglial HO-1 induction spread to engulf the whole thalamus after 11 days of TD. As in past studies, reactive iron and ferritin accumulated in microglia beginning on day 10. The lipid peroxidation product, 4-hydroxynonenal (HNE) accumulated in the remaining thalamic neurons only after 11 days of TD. These responses were not likely mediated by iNOS because HO-1 induction and HNE accumulation were comparable in iNOS knockout mice and wild-type controls. These results show that region and cell specific oxidative stress is associated with selective neurodegeneration during TD. Thus, TD is a useful model to help elucidate neuron-microglial interaction in neurodegenerative diseases associated with oxidative stress.     

CD40L deletion delays neuronal death in a model of neurodegeneration due to mild impairment of oxidative metabolism

Inflammatory/immune processes are important in the pathogenesis of neurodegenerative diseases. Thiamine deficiency (TD) models the region selective neuronal loss in brain that accompanies mild impairment of oxidative metabolism. TD induces well-defined alterations in neurons, microglia, astrocytes, and endothelial cells. To test the role of inflammatory/immune mechanisms in TD-induced neurodegeneration, the temporal profile of neurodegeneration was compared to the activation of CD68-positive microglia and ICAM-1-positive endothelial cells during TD in wild type mice and in CD40L-/- mice. CD40L-/- delayed the onset of TD-induced neuronal death as well as the activation of microglia and endothelial cells. The current results suggest that CD40L-mediated immune and inflammatory responses have a role in TD-induced neuronal death.     

Immunohistochemical estimation of rat brain somatostatin on avoidance learning impairment induced by thiamine deficiency

In rats, on the 25th day after the start of a thiamine-deficient (TD) diet, impairment of avoidance learning was significantly induced in proportion to the decrease somatostatin (SST) fluorescence intensity in the cortex, amygdala, thalamus, hypothalamus, and hippocampus, including the CA1, CA2, and dentate gyrus (DG). Only a single injection of thiamine HCl (0.5 mg/rat, subcutaneous) on the 14th day after the start of a TD diet improved the amnesia to the level of the pair-fed control and prevented the decrease in the SST level. Whereas these reversal effects of thiamine treatment were not found when the treatment was given on the 21st day after the start of a TD diet. These results indicate that, after a certain degree of thiamine deficiency, TD-induced behavioral effects might be reversible, but some neuronal fibers might be irreversibly damaged, probably due to the reduction of thiamine-dependent enzymes in brain mitochondria. The results also suggest the possibility that SST in the brain may be closely related to the avoidance learning impairment induced by TD.     

Beneficial effects of mild lifelong dietary restriction on skeletal muscle: prevention of age-related mitochondrial damage,morphological changes,and vulnerability to a chemical toxin

The effect of mild lifelong dietary restriction (DR) on age-related changes was investigated in rats. Histopathological findings were compared between 25-month-old male rats fed ad libitum and 25-month-old male rats that were calorie restricted (80% of ad libitum calories; protein 15%) from 9 weeks of age. DR-fed rats retained motor activity even in old age compared with ad libitum-fed rats. Histopathological studies on soleus muscles clarified myopathic changes in the ad libitum-fed rats, including variations in fiber size and an increase in the number of central nuclei. Increased non-grouping atrophic angulated fibers were also observed. The specimens revealed a confused arrangement of the mitochondria and decreased mitochondrial electron transduction enzyme activities, indicating mitochondrial insults in the ad libitum-fed rats. In contrast, no myopathic changes, little mitochondrial insult, and fewer angulated fibers were recognized in the DR-fed rats. The accumulations of heme oxygenase-1, crystallin, 8-hydroxydeoxyguanosine, and heat shock protein 27 were recognized in ad libitum-fed rats, indicating the attack of oxidative stress. In contrast, the expressions of these proteins were suppressed in DR-fed rats. The results suggest that even mild calorie restriction is enough to attenuate oxidative stress and age-related morphological changes in skeletal muscle. Additionally, DR was effective in protecting against methylmercury-induced pathological changes. Small fiber size and suppression of mitochondrial electron transduction enzyme activities in skeletal muscle and degenerative changes in peripheral nerves were milder in methylmercury-exposed DR-fed rats. The results indicate that mild lifelong DR also protects skeletal muscle and peripheral nerves against a chemically-induced form of oxidative stress.     

Changes in inflammatory processes associated with selective vulnerability following mild impairment of oxidative metabolism

Abnormalities in oxidative metabolism and reductions of thiamine-dependent enzymes accompany many age-related neurodegenerative diseases. Thiamine deficiency (TD) produces a cascade of events including mild impairment of oxidative metabolism, activation of microglia, astrocytes and endothelial cells that leads to neuronal loss in select brain regions. The earliest changes occur in a small, well-defined brain region, the submedial thalamic nucleus (SmTN). In the present study, a micropunch technique was used to evaluate quantitatively the selective regional changes in mRNA and protein levels. To test whether this method can distinguish between changes in vulnerable and non-vulnerable regions, markers for neuronal loss (NeuN) and endothelial cells (eNOS) and inflammation (IL-1beta, IL-6 and TNF-alpha) in SmTN and cortex of control and TD mice were assessed. TD significantly reduced NeuN and increased CD11b, GFAP and ICAM-1 immunoreactivity in SmTN as revealed by immunocytochemistry. When assessed on samples obtained by the micropunch method, NeuN protein declined (-49%), while increased mRNA levels were observed for eNOS (3.7-fold), IL-1beta (43-fold), IL-6 (44-fold) and TNF-alpha (64-fold) in SmTN with TD. The only TD-induced change that occurred in cortex with TD was an increase in TNF-alpha (22-fold) mRNA levels. Immunocytochemical analysis revealed that IL-1beta, IL-6 and TNF-alpha protein levels increased in TD brains and colocalized with glial markers. The consistency of these quantitative results with immunocytochemical measurements validates the micropunch technique. The results demonstrate that TD induces quantitative, distinct inflammatory responses and oxidative stress in vulnerable and non-vulnerable regions that may underlie selective vulnerability.     

Dietary restriction protects hippocampal neurons against the death-promoting action of a presenilin-1 mutation

Alzheimer's disease (AD) is an age-related disorder that involves degeneration of synapses and neurons in brain regions involved in learning and memory processes. Some cases of AD are caused by mutations in presenilin-1 (PS1), an integral membrane protein located in the endoplasmic reticulum. Previous studies have shown that PS1 mutations increase neuronal vulnerability to excitotoxicity and apoptosis. Although dietary restriction (DR) can increase lifespan and reduce the incidence of several age-related diseases in rodents, the possibility that DR can modify the pathogenic actions of mutations that cause AD has not been examined. The vulnerability of hippocampal neurons to excitotoxic injury was increased in PS1 mutant knockin mice. PS1 mutant knockin mice and wild-type mice maintained on a DR regimen for 3 months exhibited reduced excitotoxic damage to hippocampal CA1 and CA3 neurons compared to mice fed ad libitum; the DR regimen completely counteracted the endangering effect of the PS1 mutation. The magnitude of increase in levels of the lipid peroxidation product 4-hydroxynonenal following the excitotoxic insult was lower in DR mice compared to mice fed ad libitum, suggesting that suppression of oxidative stress may be one mechanism underlying the neuroprotective effect of DR. These findings indicate that the neurodegeneration-promoting effect of an AD-linked mutation is subject to modification by diet.     

Accumulation of amyloid precursor protein-like immunoreactivity in rat brain in response to thiamine deficiency

Thiamine deficiency (TD) is a classical model of impaired cerebral oxidation. As in Alzheimer's disease (AD), TD is characterized by selective neuronal loss, decreased activities of thiamine pyrophosphate-dependent enzymes, cholinergic deficits and memory loss. Amyloid β-protein (Aβ), a 4 kDa fragment of the β-amyloid precursor protein (APP), accumulates in the brains of patients with AD or Down's syndrome. In the current study, we examined APP and Aβ immunoreactivity in the brains of thiamine-deficient rats. Animals received thiamine-deficient diet and libitum and daily injections of the thiamine antagonist, pyrithiamine. Immunocytochemical staining and immunoblotting utilized a rabbit polyclonal antiserum against human APP^645–694 (numbering according to APP₆₉₅ isoform). Three, 6 and 9 days of TD did not appear to damage any brain region nor change APP-like immunoreactivity. However, 13 days of TD led to pathological lesions mainly in the thalamus, mammillary body, inferior colliculus and some periventricular areas. While immunocytochemistry and thioflavine S histochemistry failed to show fibrillar β-amyloid, APP-like immunoreactivity accumulated in aggregates of swollen, abnormal neurites and perikarya along the periphery of the infarct-like lesion in the thalamus and medial geniculate nucleus. Immunoblotting of the thalamic region around the lesion revealed increased APP-like holoprotein immunoreactivity. APP-like immunoreactive neurites were scattered in the mammillary body and medial vestibular nuclei where the lesion did not resemble infarcts. In the inferior colliculus, increased perikaryal APP-like immunostaining occurred in neurons surrounding necrotic areas. Regions without apparent pathological lesions showed no alteration in APP-like immunoreactivity. Thus, the oxidative insult associated with cell loss, hemorrhage and infarct-like lesions during TD leads to altered APP metabolism. This is the first report to show a relationship between changes in APP expression, oxidative metabolism and selective cell damage caused by nutritional/cofactor deficiency. This model appears useful in defining the role of APP in the response to central nervous system injury, and may also be relevant to the pathophysiology of Wernicke-Korsakoff syndrome and AD.     

Loss of astrocytic glutamate transporters in Wernicke encephalopathy

Wernicke encephalopathy (WE), a neurological disorder caused by thiamine deficiency (TD), is characterized by structural damage in brain regions that include the thalamus and cerebral cortex. The basis for these lesions is unclear, but may involve a disturbance of glutamatergic neurotransmission. We have therefore investigated levels of the astrocytic glutamate transporters EAAT1 and EAAT2 in order to evaluate their role in the pathophysiology of this disorder. Histological assessment of the frontal cortex revealed a significant loss of neurons in neuropathologically confirmed cases of WE compared with age‐matched controls, concomitant with decreases in α‐internexin and synaptophysin protein content of 67 and 52% by immunoblotting. EAAT2 levels were diminished by 71% in WE, with levels of EAAT1 also reduced by 62%. Loss of both transporter sites was confirmed by immunohistochemical methods. Development of TD in rats caused a profound loss of EAAT1 and EAAT2 in the thalamus accompanied by decreases in other astrocyte‐specific proteins. Treatment of TD rats with N‐acetylcysteine prevented the downregulation of EAAT2 in the medial thalamus, and ameliorated the loss of several other astrocyte proteins, concomitant with increased neuronal survival. Our results suggest that (1) loss of EAAT1 and EAAT2 glutamate transporters is associated with structural damage to the frontal cortex in patients with WE, (2) oxidative stress plays an important role in this process, and (3) TD has a profound effect on the functional integrity of astrocytes. Based on these findings, we recommend that early treatment using a combination of thiamine AND antioxidant approaches should be an important consideration in cases of WE. © 2009 Wiley‐Liss, Inc.     

Downregulation of complexin I and complexin II in the medial thalamus is blocked by N-acetylcysteine in experimental Wernicke's encephalopathy

Metabolic dysfunction as a consequence of thiamine (vitamin B1) deficiency (TD), a model of Wernicke's encephalopathy, leads to elevation of extracellular glutamate concentration in vulnerable brain regions consistent with the development of excitotoxicity. Complexin I and complexin II are two genes labeling principally inhibitory and excitatory synapses, respectively. Because current evidence supports an important role for complexins in the modulation of neurotransmitter release, we examined the involvement of both proteins in the pathology of the medial thalamus and inferior colliculus in TD rats by immunoblotting. At the symptomatic stage, complexin I and complexin II levels in the medial thalamus were decreased by 63% and 45%, respectively, compared to control animals, but were unchanged in the inferior colliculus. These changes in thalamus were also observed using immunohistochemical methods, and seemed to be due to downregulation of both proteins because synaptophysin levels were unaffected in this brain region. In addition, cotreatment with the antioxidant N- acetylcysteine prevented both neuronal loss and downregulation of complexins. Our findings suggest dysregulation of excitatory and inhibitory neurotransmitter release in the medial thalamus, which is not present in the inferior colliculus. Furthermore, loss of complexin I and II in the thalamus may be mediated by processes that involve oxidative stress. Such changes in complexin levels may contribute to the pathophysiology of thalamic damage in TD, and offer a potential basis for the well-known differences in pathology between this structure and the inferior colliculus in this disorder.     

Dietary restriction to accompany the aging process in mice Can it be neuroprotective?

BACKGROUND: Prophylactic dietary restriction (DR), whether lifelong or started in adulthood,retards the aging process and attenuates cognitive decline in rodents. However, whether the anti-aging and neuroprotective efficacy of DR initiate late in life or accompany the aging process remains unclear.OBJECTIVE: The present study sought to: (1) determine if DR could protect against behavioral decline in mice when implemented during the aging process induced by D-galactose and (2) examine neuronal apoptosis in these aged brains and whether DR could block apoptosis.DESIGN, TIME AND SETTING: The randomized controlled animal study. The experiment was performed at the Experimental Animal Center of Capital Medical University and the Laboratory Center of School of Public Health of Captial Medical University of China from April 2006 to October 2007.MATERIALS: D-galactose (D-gal) was purchased from Beijing Chemical-Regent Company (Beijing, China). Terminal transferase dUTP nick end labeling (TUNEL) detection kit was obtained from Roche, Germany. Assay kits for antioxidant enzyme activities and malondialdehyde contents were purchased from Jiancheng Institute of Biotechnology (Nanjing, China). Morris water maze (Friends Honesty Life Sciences Co. Ltd., Hong Kong, China) and Flow Cytometry (Coulter, USA) were used in this study.METHODS: A total of 40 male Institute of Cancer Research (lCR) mice, 3 months old, were equally and randomly divided into D-gal treatment, DR treatment, D-gal + DR treatment and normal control groups, and were then randomly assigned to one of two feeding regimens: ad libitum access to food or DR which received a 70% amount of daily food intake as that by ad libitum fed mice. There were two replicates per feeding regimen and mice were fed for 10 weeks,with or without a daily subcutaneous injection of D-gal at 100 mg/kg.MAIN OUTCOME MEASURES: Animals' spatial learning and memory performance were tested in the Morris water maze. Neuronal apoptosis rates were evaluated by Annexin V/flow cytometry assay and TUNEL assay. Lipid peroxidation levels and antioxidant defense capacity of the brain were measured using testing kits.RESULTS: DR markedly reduced the prolonged escape latency of D-gal mice in the water maze test (P＜0.01). Annexin V and TUNEL assays showed that the D-gal mice had a significant higher percentage of neuronal apoptosis compared with normal control mice (P＜0.05), and that DR treatment markedly decreased this apoptotic cell death (P＜0.05). DR also reversed the decline of total superoxide dismutase and glutathione peroxidase activities and the increase of malondialdehyde levels in the brain of D-gal mice (P＜0.05, respectively).CONCLUSION: DR reduces the impact of D-gal-induced brain aging in mice and can reverse performance decline and neurobiochemical impairments. These results demonstrate that implementation of DR in conditions of chronic oxidative stress can be neuroprotective, and that senium DR can be beneficial for healthy aging.     

N-acetylcysteine attenuates early induction of heme oxygenase-1 following traumatic brain injury

Traumatic brain injury (TBI) results in a cascade of events that includes the production of reactive oxygen species. Heme oxygenase-1 (HO-1) is induced in glial cells following head trauma, suggestive of oxidative stress. We have studied the temporal and spatial effects of the antioxidant N-acetylcysteine (NAC) on HO-1 levels following lateral fluid-percussion injury by immunoblotting and immunohistochemistry. In the injured cerebral cortex, maximal HO-1 induction was seen 6 h post-TBI and was maintained for up to 24 h following the insult, while the ipsilateral hippocampus and thalamus showed marked induction at 24 h postinjury. In all three brain regions, little or no HO-1 immunoreactivity was observed on the contralateral side. Astrocytes exhibited positive immunoreactivity for HO-1 in the injured cerebral cortex, hippocampus, and thalamus, while some neurons and microglia were also immunoreactive in the injured cortex. The administration of NAC 5 min following TBI resulted in a marked reduction in this widespread induction of HO-1, concomitant with a decrease in the volume of injury in all three brain regions. Together, these findings indicate that HO-1 induction is related to both oxidative and injury characteristics of the affected tissue, suggesting that protein expression of this gene is a credible marker of oxidative damage in this model of TBI.     

硫胺素缺乏引起脑内β淀粉样蛋白沉积和tau蛋白磷酸化的增加

目的：探讨硫胺素缺乏（TD）对脑内β淀粉样蛋白（Aβ）沉积、tau蛋白磷酸化的影响。方法：硫胺素剥夺饮食结合腹腔注射吡啶硫胺制作TD小鼠模型,正常对照组给予正常饮食及腹腔注射生理盐水。造模13d后取脑,行苏木精-伊红染色观察两组小鼠脑内易损区域病理改变,免疫组织化学染色检测脑内Aβ沉积、tau蛋白磷酸化及β-分泌酶（BACE）的表达情况。结果：苏木精-伊红染色显示TD模型组小鼠内侧丘脑出现典型的对称性针尖样出血;免疫组化显示TD模型组小鼠皮质、海马及丘脑均出现Aβ沉积,且丘脑Aβ沉积较皮质及海马更为明显,tau蛋白磷酸化及BACE的阳性细胞数显著增加。正常对照组小鼠苏木精-伊红染色脑内未见病理损伤,皮质、海马及丘脑均未发现Aβ沉积,tau蛋白磷酸化及表达BACE的阳性细胞数均明显低于TD模型组,两组差异有统计学意义（P〈0．05）。结论：TD可引起Aβ沉积、tau蛋白磷酸化增加等阿尔茨海默病的特征性病理改变,且Aβ沉积可能与上调BACE的表达有关。     

Heme Oxygenase in the Experimental ALS Mouse

Heme oxygenase-1 (HO-1) is a stress protein inducible in some cells by oxidative stress. The status of heme oxygenase was investigated in a transgenic mouse model of amyotrophic lateral sclerosis (ALS) since oxidative mechanisms are postulated in neuronal injury. Three ALS mice [(SOD1-G93A)1Gur] and three controls [(SOD-1)2Gur] were obtained from The Jackson Laboratory. Behavioral differences suggestive of neurodegeneration in ALS mice developed at 4–5 months of age. All mice were killed at 7–8 months of age. Tissue vacuolation, cell loss, and the presence of GFAP⁺cells were noted in the spinal cords of ALS mice. Spinal cord motor neurons in both control and ALS mice stained positive for heme oxygenase-2 (HO-2). While not precluding the presence of low levels of HO-1 neither immunohistochemical staining nor Western blot analysis provided evidence for significant HO-1 induction in degenerating spinal cord.     

Thiamine deficiency during pregnancy leads to cerebellar neuronal death in rat offspring: role of voltage-dependent K+ channels

Oxidative stress, selective neuronal loss, and diminished activity of thiamine-dependent enzymes play a role in many neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease and Huntington's disease. To further understand the major implications of thiamine deficiency (TD) in neuronal death, we induced TD during pregnancy and evaluated the effects on the offspring. The body and brain weights of pups from thiamine-deficient dams were significantly smaller than normal. Loss of neuronal viability was examined by trypan blue exclusion assay, and demonstrated increased cytotoxicity in primary cultures of TD neurons. Additionally, cerebellar cultures were exposed to thiamine-free cell culture medium to better explore the effects of thiamine withdrawal. Alterations in potassium current has previously been associated with the development of cell death. In this study, we examined the TD effects on delayed rectifier and A-type K+ channels, two well-known voltage-activated K+ channels involved in the regulation of action potential firing in cerebellar granule neurons. Current recordings were performed in cultured rat cerebellar granule neurons at day 7, using the whole-cell voltage-clamp technique. Our data demonstrate that thiamine deficiency provoked a significant decrease in the voltage-dependent K+ membrane conductance. Finally, TD markedly depressed the transient A-type K+ currents.     

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

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

Dietary restriction and 2-deoxyglucose administration improve behavioral outcome and reduce degeneration of dopaminergic neurons in models of Parkinson's disease.

Parkinson's disease (PD) is an age-related disorder characterized by progressive degeneration of dopaminergic neurons in the substantia nigra (SN) and corresponding motor deficits. Oxidative stress and mitochondrial dysfunction are implicated in the neurodegenerative process in PD. Although dietary restriction (DR) extends lifespan and reduces levels of cellular oxidative stress in several different organ systems, the impact of DR on age-related neurodegenerative disorders is unknown. We report that DR in adult mice results in resistance of dopaminergic neurons in the SN to the toxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). MPTP-induced loss of dopaminergic neurons and deficits in motor function were ameliorated in DR rats. To mimic the beneficial effect of DR on dopaminergic neurons, we administered 2-deoxy-D-glucose (2-DG; a nonmetabolizable analogue of glucose) to mice fed ad libitum. Mice receiving 2-DG exhibited reduced damage to dopaminergic neurons in the SN and improved behavioral outcome following MPTP treatment. The 2-DG treatment suppressed oxidative stress, preserved mitochondrial function, and attenuated cell death in cultured dopaminergic cells exposed to the complex I inhibitor rotenone or Fe2+. 2-DG and DR induced expression of the stress proteins heat-shock protein 70 and glucose-regulated protein 78 in dopaminergic cells, suggesting involvement of these cytoprotective proteins in the neuroprotective actions of 2-DG and DR. The striking beneficial effects of DR and 2-DG in models of PD, when considered in light of recent epidemiological data, suggest that DR may prove beneficial in reducing the incidence of PD in humans.