Thiamine Deficiency Effects And Mechanisms Of Beta-amyloid Precursor Protein Processing And A¦Â Generation

Alzheimer's disease(AD)is the most common cause of dementia in the elderly worldwide.Neuropathological hallmarks of AD are extracellular senile plaques(SPs), intracellular neurofibrillary tangles(NFTs)and massive neuronal loss in vulnerable brain regions.The pathogenesis of AD is still poorly understood and current available treatments for AD are unsatisfactory.β-amyloid peptide(Aβ)is the major component of SPs,which is derived from proteolytic processing ofβ-amyloid precursor protein (APP).Mounting evidence suggests that Aβplays an initial role in AD pathogenesis, Therefore,unraveling the molecular mechanism of Aβmetabolism is important to AD pathogenesis research.Thiamine(Vitamin B₁)is an essential nutrition factor for human beings.The active form of thiamine in vivo is thiamine pyrophosphate(TPP).TPP acts as a co-enzyme of thiamine-dependent enzymes,which include the pyruvate dehydrogenase complex(PDHC),α-ketoglutarate dehydrogenase complex(KGDHC) and transketolase(TK).Thiamine is also essential to keep the cellular redox state and glutathione reductase activity.Thiamine deficiency(TD)impairs the activities of thiamine-dependent enzymes which result in dysfunction of tricarboxylic acid cycle and shortage of adenosine-triphosphate(ATP).Simultaneously,the accumulation of reactive oxygen species(ROS)leads to severe oxidative impairment during TD process.The nervous system is highly sensitive to TD,which may cause a serious neurological disorder called Wernicke-Korsakoff's syndrome(WKS).The clinical features of WKS include confusion,cognitive deficits,memory loss,and,at late stage of WKS,enlarged ventricle and neuronal loss are also found.The activity of KGDHC decreases in WKS patient brain.In brains of TD animal models,common features of neurodegenerative diseases can be found,such as selective neuronal loss,activation and proliferation of microglia and astrocytes cells.Furthermore,it is reported that TPP and the activities of thiamine-dependent enzymes both decline in AD patients, which suggests that TD may contribute to the AD pathology.However,the exact effect(s)of TD in AD pathogenesis is still unclear.In present work,we investigated the effect(s)of TD on APP processing using cultured Swedish mutant APP(APPsw)transfected SH-SY5Y cells and C57BL/6J mouse with a series of biochemical and morphological techniques.Firstly,in APPsw stably transfected SH-SY5Y cells,TD caused moderate cell death(about 20%)after 5-days treatment,while the numbers of apoptotic cells elevated by 2-fold and the cell viability decreased by 50%.Secondly,the amounts of Aβ,both secreted and intracellular forms were elevated during TD:After 5 days TD treatment,the basic secreted(5.52±0.12 pg/mg)and intracellular(23.4±5.17 pg/mg) Aβ₄₀were elevated to 18.57±2.31 pg/mg and 86.03±8.19 pg/mg,respectively.The basic secreted(0.66±0.23 pg/mg)and intracellular(7.06±1.03 pg/mg)Aβ₄₂were elevated to 3.85±0.52 pg/mg and 23.52±1.02 pg/mg,respectively.Thirdly,during the TD process,the mature from of APP is decreased and the immature from is unchanged.Therefore,Aβaccumulation should not be derived from increased expression of its precursor protein.Fourthly,the activity ofβ-secretase was up regulated and the activity ofγ-secretase is unchanged during TD.Real-time polymerase chain reaction(Real time-PCR)and immunoblotting assay showed that the expression ofβ-site APP cleaving enzyme 1(BACE1)gene is unaltered,and, however,the maturation of BACE1 increased,the mature BACE1 increased by about 2-fold,and immature BACE1 decreased by about 50%.This change may result in an elevatedβ-secretase activity.To further confirm the changes ofβ-secretase activity, we examined the level ofβ-C terminal fragment(β-CTF)of APP,the product ofβ-secretase cleavage.The level ofβ-CTF was elevated during TD.Fifthly,the above abnormal alteration induced by TD can be restored with thiamine re-supplement for 24 hours after 5 days TD treatment,implying that the effectiveness of thiamine re-supplement after TD.Sixthly,addingβ-secretase inhibitor to the 5-day TD treated culture medium could efficiently inhibit the accumulation of Aβandβ-CTF. Seventhly,5-day TD treatment increased ROS by nearly 3-fold compared with the control groups,which can be significantly attenuated by antioxidant Troiox.Trolox also can inhibit the TD induced Aβaccumulation and maturation of BACE1.Finally, synthetic Aβ_1-40exposure(250pmol/L)during TD treatment can elevate the ROS level significantly compared with TD treatment only,indicating that Aβaccumulation enhanced the oxidative stress caused by TD.These findings on TD cell model were also confirmed in vivo in TD mouse model: (1)neuronal loss was found in submedial thamalic nucleus(SmTN)after 9-days TD treatment;(2)dramatic Aβaccumulation was detected in thalamus region:the Aβ₄₀ level of control group and TD 10-days group is 0.97±0.15 and 3.34±0.33 pmol/g, respectively,and the Aβ₄₂level of control group and TD 10-days group is 0.16±0.01 and 0.49±0.02 pmol/g;(3)restoration with thiamine after 8-days TD treatment can reduce the Aβ₄₂accumulation caused by TD.Taken together,our results imply that:(1)the oxidative stress caused by TD promotes the amyloidogenic APP processing and resulting in Aβaccumulation;(2) the oxidative stress up-regulates theβ-secretase activity though enhancing the BACE1 maturation;(3)conversely,Aβaccumulation exacerbates the oxidative stress during TD treatment,suggesting that there may be a positive feedback cycle between Aβaccumulation and oxidative stress.We have found that TD can cause oxidative stress and promote the amyloidogenic APP processing,and resulting in Aβaccumulation.Furthermore,a positive feedback cycle exists between Aβaccumulation and oxidative stress.These effects may play important roles in AD pathogenesis and/or progression.Finally,our thiamine re-supplement experiment results showed that it may be effective and important to supply thiamine for treatment of the AD patients who were proved to be TD.