Thiamine Deficiency And Pyrithiamine Increase β-amyloid Accumulation, Tau Hyperphosphorylation, And Activities Of Glycogen Synthase Kinase-3 In Brains

Alzheimer's disease is the most prevalent neurodegenerative disorder marked by memory loss and cognitive impairment that occurred mainly in elder people. The drastic disturbance of glucose metabolism in brain is one of the striking features in Alzheimer's disease (AD).The impairments of cerebral glucose utilization and energy metabolism in the patients with AD are very early features that precede or accompany the initial stage of cognitive impairment. Animal experiments also suggest that abnormality of cerebral glucose metabolism can be the key etiological factor. Declined energy metabolism induces dysfunction of brain by injuring mitochondric function, which triggers generation of reactive oxygen species. Thiamine is critical for glucose metabolism of brain as a cofactor of several rate-limited enzymes of Kreb cycle and pentose phosphate pathway (PPP), which are two predominant pathways for glucose metabolism in mammalian brain. These enzymes include transketolase in the non-oxidative branch of the PPP, andα-ketoglutarate dehydrogenase complex (KGDHC) and pyruvate dehydrogenase complex (PDHC) in Kreb cycle. Thiamine deficiency (TD) leads to the dysfunction of Kreb cycle and the ppp, resulting in the disturbance of cerebral glucose and energy metabolism. Epidemiological investigations have also demonstrated that elderly people as well as AD patients have prevalently sub-clinical TD.The levels of serum thiamine and its phosphate ester derivatives in the patients with AD remarkably decrease compared with health control subjects. The activities of serum transketolase, KGDHC, and PDHC are diminished by 50% or more in AD.In addition, TD can cause selective neuronal death, inflammation, and glial abnormal activation,the common features of many aging-related neurodegenerative diseases including AD. TD increases the activity of brainβ-secretase and oxidative stress as well as exacerbates cerebralβ-amyloid (Aβ) deposition in APP/PS1 transgenic mice. Furthermore, the phosphorylated Tau level in CSF is elevated in patients with TD. Thus, we consider that TD or abnormal metabolism of thiamine and reduced thiamine-dependent enzymes activities may play an important role in development of AD and directly involve in the characterized AD pathological changes such as Aβdeposition and Tau hyperphosphorylation. Moreover, researchers have found that sub-clinical thiamine deficiency in elderly people and AD patients is not linked to the decline of absorption function or co-morbidity. What causes such situation? Currently, the researchers make thiamine deficiency model by the combination of dietary thiamine deprivation and thiamine antagonist treatment. We have known that elderly people seldom have dietary thiamine deficiency, does it mean that we can hypothesis such situation is caused by the existing of some unknown thiamine antagonists? With this question, we treat mice seperately with dietary thiamine deficiency and thiamine antagonist to compare their influence on the memory and AD-like pathological changes in APP/PS1 transgenic mice and wild-type mice.In addition, the effect of TD and pyrithiamine on glycogen synthase kinase-3 is investigated.Part 1:Influence of pyrithiamine on memory and pathologic alterations in APP/PS1 transgenic miceObjective:To explore the influence of pyrithiamine and thiamine deficiency on the memory and the pathological changes such as brain Aβdeposition and Tau hyperphosphorylation in APP/PS1 transgenic mice.Methods:Twenty-week-old APP/PS1 transgenic mice were divided into four groups randomly:control group (normal diet+NS/d i.p.abbreviated as Ctrl group below), low-dose group of pyrithiamine (normal diet+pyrithiamine 250μg/kg/d i.p. abbreviated as PT250 group below), high-dose group of pyrithiamine (normal diet+ pyrithiamine 500μg/kg/d i.p. abbreviated as PT500 group below) and dietary thiamine deprivation group (thiamine deficiency diet+NS/d i.p. abbreviated as TD group below).Mice of each group were intraperitoneally administrated daily with the above drugs for four weeks. At the end of the period, all mice were subjected to observe cognitive testing by Morris water maze. After Morris water maze, mice were sacrificed. Brains of them were taken out after perfusion and postfixed in 4% paraformaldehyde overnight, followed by embedded with paraffin. The paraffin sections were used to observe brain Aβdeposition and Tau hyperphosphorylation.Results:The probe trials were conducted 24h after the last training trial and the ratio of the time spent in target (IV) quadrant was calculated. The ratio of Ctrl group was 25.00±3.40%,PT250 group was 28.29±3.44%,PT500 group was 23.38±3.54%,TD group was 20.30±4.97%.APP/PS1 transgenic mice in PT500 group and TD group spent less time in IV quadrant compared with that of Ctrl group, but showed no significant difference. Pathologically, the number of amyloid plaques in the brains of APP/PS1 Ctrl group was 45.50±3.84, APP/PS1 PT250 group was 77.25±6.00, APP/PS1 PT500 group was 73.00±9.97, and APP/PS1 TD group wasl31.5±12.23. The results showed the numbers of brain amyloid plaques in APP/PS1 PT250, PT500, and TD groups were significantly more than that of APP/PS1 Ctrl group (P<0.001 for PT250, PT500, and TD groups vs.Ctrl group). The number of the p-Tau positive cells in the brains of Ctrl group was 156.62±12.18,PT250 group was 204.30±25.27, PT500 group was 272.88±44.60,and TD group was 268.28±24.29.The numbers of brain p-Tau positive cells in APP/PS1 PT250, PT500, and TD groups were significantly more than that of APP/PS1 Ctrl group (P< 0.05 for PT250 and 500 groupsvs. Ctrl group; P<0.01 for TD group vs. Ctrl group).Conclusion:pyrithiamine and diet-induced thiamine deficiency have not much influence on the memory of APP/PS1 transgenic mice, but significantly aggravated the AD-related pathological alterations in APP/PS1 transgenic mice.Part 2 Influence of pyrithiamine on memory and pathologic alterations in wild-type miceObjective:To observe the influence of pyrithiamine and diet-induced thiamine deficiency on the memory and the pathological changes of wild-type mice.Methods:Twenty-week-old wild-type mice were divided into four groups randomly: Ctrl group (normal diet+NS/d i.p. abbreviated as Ctrl group below), low-dose group of pyrithiamine (normal diet+pyrithiamine 250μg/kg/d i.p. abbreviated as PT250 group below), high-dose group of pyrithiamine (normal diet+pyrithiamine 500μg/kg/d i.p. abbreviated as PT500 group below) and dietary thiamine deprivation group (thiamine deficiency diet+NS/d i.p. abbreviated as TD group below).The mice of each group were intraperitoneally administrated daily with the above drugs for four weeks. At the end of the period, all mice were subjected to observe cognitive testing by Morris water maze. After Morris water maze, mice were sacrificed. Then, the brains were taken out after perfusion and postfixed in 4% paraformaldehyde overnight, followed by embedded with paraffin. The paraffin sections were used to observe brain Aβdeposition and Tau hyperphosphorylation. Amyloid plaques were not obvious in wild-type mice, we used Aβ1-42 ELISA assay to evaluate the quantity ofAβ.Results:In probe trials, the ratio of the time spent in target (IV) quadrant, the ratio of Ctrl group was 38.28±4.52%,PT250 group was 24.38±2.98%,PT500 group was 21.36±3.62%,TD group was 15.41±2.14%. The results showed that pyrithiamine and TD significantly impaired the memory function of the wild-type mice (PT250 vs. Ctrl group P<0.05;PT500, TD vs. Ctrl group P<0.01).Furthermore, the number of p-Tau positive cells in the brains of Ctrl group was 131.22±8.98,PT250 group was 155.58±9.59,PT500 group was 178.27±9.83,and TD group was 171.69±8.52.The results showed that the numbers of brain p-Tau positive cells in PT500, and TD groups were significantly more than that of control group (P< 0.05 for PT500 group and P<0.01 for TD group vs. control group).Amyloid plaque was not observed in wild-type mice. However, in Aβ1-42 ELISA assay, we found the increased level of brain Aβ1-42 significantly in groups of pyrithiamine administration and TD treatment: the content of brain Aβ1-42 in Ctrl group was 406.86±18.17 pg/mg,PT250 group was 493.02±25.99pg/mg,PT500 group was 582.58±22.91pg/mg,TD group was 602.38±20.09pg/mg (P< 0.05 for PT250 vs. control group; P<0.001 for PT500 group and TD group vs. control group).Conclusion:pyrithiamine and diet-induced thiamine deficiency impair the memory and aggravate the pathological alterations significantly in wild-type mice.Part 3 Effects of thiamine deficiency and pythiamine on the activities of GSK-3in brains of wild-type miceObjective:To explore the effects of thiamine deficiency and pyrithiamine on the activities of brain GSK-3 in wild-type mice.Methods:Wild-type mice brain tissues were taken out from-80℃and divided into two parts for the use of western blot and enzyme activity testing. We compared the changes of the ratio of phospho-GSK-3a/GSK-3a and phospho-GSK-3p/GSK-3βby western blot. The activities of GSK-3,GSK-3a, and GSK-3βwere determined by kits according to the manufacturer's instructions.Results:Western blot results showed the ratio of p-GSK-3a/GSK-3a in Ctrl group was 1.445±0.114, PT250 group was 1.454±0.071,PT500 group was 1.225±0.245,and TD group was 0.879±0.120 (P<0.05 for TD group vs. Ctrl group). PT500 and TD groups showed the decreased ratios of phospho-GSK-3β(Ser9)/GSK-3βwith the following specific values:Ctrl group 0.6154±0.089;PT250 group 0.573±0.121; PT500 group 0.348±0.099; TD group 0.298±0.060 (P<0.05 for PT500 group vs. Ctrl group; TD<0.01 for TD group vs. Ctrl group).Furthemore, we found that pyrithiamine at PT500 and TD groups significantly increased the enzyme activities of total GSK-3,GSK-3a and GSK-3p.Conclusion:pyrithiamine treatment and dietary-induced thiamine deficiency increased the activities of GSK-3.It indicates that GSK-3 may be involved in the pathologic mechanism of TD...