Down-regulation amyloid β-protein 42 production by interfering with transcript of presenilin 1 gene with siRNA

AIM:ToinvestigatethepathogenesisofAβ42yieldingandnewdrugtargetsaswellasthepossibilityofRNAinterference(RNAi)techniquefortreatmentofAlzheimerdisease(AD).METHODS:HumanADpresenilin1(PS1)cDNAsequencewasobtainedfromNCBIwebsite.ThethreesitesofRNAiactionandonemissensecontrolsitewereselectedinPS1cDNAthroughonlinedesignofAmbioncompany.Toconfirmspecificityofthesesites,weconductedaBLASTsearchoftheIMAGEESTlibrary.Thecorrespondingdouble-strandedDNAwasusedtoconstructpSilencer3.1-H1p…     

AD治疗新药物——γ-分泌酶抑制剂

家族性阿尔采末病属常染色体显性遗传 ,其中大部分与编码早老素的基因发生突变所导致的γ 分泌酶功能异常有关 ,γ 分泌酶是一包括PS二聚体、Nicastrin、PEN 2等组分的多酶复合体。γ 分泌酶剪切APP、Notch、E cadherin、ErbB 4受体酪氨酸激酶等膜蛋白。目前已发现了许多类型的γ 分泌酶抑制剂 :基于二氟酮、二氟乙醇基团的γ 分泌酶抑制剂 ;脲多肽模拟化合物 ;基于羟乙基二肽电子等配体的化合物 ;具有α 螺旋结构的小肽 ;具有 4 氯 异香豆素基本母核的非肽抑制剂 ;含有丙氨酰基团的化合物。但这些抑制剂目前大多还只是作为一种工具药来研究γ 分泌酶的结构、功能以及作用机制。其中选择性抑制Aβ生成的γ 分泌酶抑制剂很有希望成为一种高效的阿尔采末病治疗药物     

Targeting 13-secretase with RNAi in neural stem cells for Alzheimer＇s disease therapy

There are several major pathological changes in Alzheimer＇s disease, including apoptosis of cho- linergic neurons, overactivity or overexpression of 13-site amyloid precursor protein cleaving enzyme 1 （BACE1） and inflammation. In this study, we synthesized a 19-nt oligonucleotide targeting BACE1, the key enzyme in amyloid beta protein （AI3） production, and introduced it into the pSilenCircle vector to construct a short hairpin （shRNA） expression plasmid against the BACE1 gene. We transfected this vector into C17.2 neural stem cells and primary neural stem cells, resulting in downregulation of the BACE1 gene, which in turn induced a considerable reduction in reducing AI3 protein production. We anticipate that this technique combining cell transplantation and gene ther- apy will open up novel therapeutic avenues for Alzheimer＇s disease, particularly because it can be used to simultaneously target several pathogenetic changes in the disease.     

阿尔茨海默病重要分泌酶BACE1及其抑制剂

阿尔茨海默病（AD）是一种最为普遍的痴呆。其病理特征是神经细胞外不溶性淀粉样蛋白Aβ以及胞内由过磷酸化tau形成的纤维缠结。这种胞外聚合物主要由Aβ4两组成,它是由淀粉样前体蛋白APP依次经β分泌酶（β-secretase）和γ分泌酶（γ～secretase）剪切所产生的。因此,通过抑制此两种酶很有可能能够减少Aβ的产生从而延缓病程。由于γ分泌酶有众多重要的生理功能,被抑制后会产生很多较为严重的副作用,因此β分泌酶的抑制剂可能是对抗AD药物研发的更有效切入点。本文主要对影响BACE1的表达及活性的相关因素和最新研制成功的β分泌酶抑制剂做简要综述。     

Advances in the study of Alzheimer＇s disease

Alzheimer＇ s disease （AD） is the most common cause of dementia, and the only treatment currently available for the disease is acetylcholinesterase inhibitors. Recent progress in understanding the molecular and cellular pathophysiology of Alzheimer＇s disease has suggested possible pharmacological interventions, including acetylcholineseterase inhibitors; secretase inhibitors; cholesterol lowering drugs; metal chelators and amyloid immunization. The objective of this paper is to review the main drugs possibly used for AD and their future therapeutic effects.     

Cytoplasmic domain of the beta-amyloid protein precursor of Alzheimer's disease: function, regulation of proteolysis, and implications for drug development

The beta-amyloid protein precursor (APP) has been extensively studied for its role in amyloid production and the pathogenesis of Alzheimer's disease (AD). However, little is known about the normal function of APP and its biological interactions. In this Mini-Review, the role of the cytoplasmic domain of APP in APP trafficking and proteolysis is described. These studies suggest that proteins that bind to the cytoplasmic domain may be important targets for drug development in AD.     

Identity and function of gamma-secretase

gamma-Secretase catalyzes intramembrane proteolysis of various type I membrane proteins, including the amyloid-beta precursor protein and the Notch receptor. Despite its importance in the pathogenesis of Alzheimer's disease and to normal development, this protease has eluded identification until only very recently. Four membrane proteins are now known to be members of the protease complex: presenilin, nicastrin, aph-1, and pen-2. Recent findings suggest that these four proteins are sufficient to reconstitute the active gamma-secretase complex and that together they mediate the cell surface signaling of a variety of receptors via intramembrane proteolysis.     

Neuronal and glial beta-secretase (BACE) protein expression in transgenic Tg2576 mice with amyloid plaque pathology

We measured tissue distribution and expression pattern of the beta-site amyloid precursor protein (APP)-cleaving enzyme (BACE) in the brains of transgenic Tg2576 mice that show amyloid pathology. BACE protein was expressed at high levels in brain; at lower levels in heart and liver; and at very low levels in pancreas, kidney, and thymus and was almost absent in spleen and lung when assayed by Western blot analysis. We observed strictly neuronal expression of BACE protein in the brains of nontransgenic control mice, with the most robust immunocytochemical labeling present in the cerebral cortex, hippocampal formation, thalamus, and cholinergic basal forebrain nuclei. BACE protein levels did not differ significantly between control and transgenic mice or as a result of aging. However, in the aged, 17-month-old Tg2576 mice there was robust amyloid plaque formation, and BACE protein was also present in reactive astrocytes present near amyloid plaques, as shown by double immunofluorescent labeling and confocal laser scanning microscopy. The lack of astrocytic BACE immunoreactivity in young transgenic Tg2576 mice suggests that it is not the APP overexpression but rather the amyloid plaque formation that stimulates astrocytic BACE expression in Tg2576 mice. Our data also suggest that the neuronal overexpression of APP does not induce the overexpression of its metabolizing enzyme in neurons. Alternatively, the age-dependent accumulation of amyloid plaques in the Tg2576 mice does not require increased neuronal expression of BACE. Our data support the hypothesis that neurons are the primary source of beta-amyloid peptides in brain and that astrocytic beta-amyloid generation may contribute to amyloid plaque formation at later stages or under conditions when astrocytes are activated.     

TMP21 degradation is mediated by the ubiquitin‐proteasome pathway

The presenilin‐associated complex regulates two independent intramembranous cleavage activities, i.e. γ‐secretase and ε‐secretase activity. The γ‐secretase complex requires four critical components for its activity: presenilin 1, anterior pharynx‐defective 1, nicastrin 1 and presenilin enhancer 2, all of which are degraded through the ubiquitin‐proteasome pathway. Recently, TMP21, a type I transmembrane protein involved in endoplasmic reticulum/Golgi transport, was identified as a member of the presenilin complex. Knockdown of TMP21 selectively regulated pathogenic γ‐secretase activity, resulting in increased amyloid β protein 40 and 42, without affecting the ε‐cleavage of Notch. A further understanding of TMP21 degradation is required to examine the biological consequences of TMP21 protein level aberrations and their potential role in the pathogenesis of Alzheimer’s disease and drug development. Here we show that human TMP21 has a short half‐life of approximately 3 h. Treatment with proteasomal inhibitors can increase TMP21 protein levels in both a time‐ and dose‐dependent manner, and both co‐immunoprecipitation and immunofluorescent staining show that TMP21 is ubiquitinated. Inhibition of the lysosomal pathway failed to show a dose‐dependent increase in TMP21 protein levels. Taken together, these results indicate that the degradation of TMP21, as with the other presenilin‐associated γ‐secretase complex members, is mediated by the ubiquitin‐proteasome pathway.     

The discovery of novel β-secretase inhibitors: pharmacophore modeling, virtual screening, and docking studies

This article describes the identification of two small molecular inhibitors for β-secretase by integrating virtual screening with fluorescence resonance energy transfer bioassay. A ligand-based pharmacophore model was developed, and the sequential virtual screening of ZINC database was performed using the acquired pharmacophore model and molecular docking. Biological evaluation of 10 virtual hits led to the identification of two novel inhibitors with IC(50) values of 4.76 and 0.31 μm, respectively. These two moderate inhibitors could represent new potentials for the development of anti-Alzheimer's disease agents.