Advances in understanding the β-amyloid precursor protein intracellular domain

老年性痴呆症(Alzheimer's disease,AD)一个重要的病理学特征,是在神经细胞外形成由β-淀粉样蛋白(β-amyloid,Aβ)组成的淀粉样斑(amyloid plaques)。β-淀粉样蛋白前体蛋白(β-amyloid procursor protein,APP)经β-分泌酶和γ-分泌酶依次水解后产生Aβ和APP胞内结构域(APP intracellular domain,AICD)。现在已经知道Aβ在AD的发病机制中起着关键作用,但是关于AICD的生理及病理功能还不清楚。近年来研究发现AICD可以与细胞内多种蛋白相互作用,而且AICD在基因转录、细胞凋亡以及APP的加工和运输过程中均有调节功能。本文针对这一领域的研究进展,对AICD的生理及病理功能进行探讨。One of the neuropathologic hallmarks of Alzheimer’s disease (AD) is the presence of senile plaques which consist of β-amyloid peptide (Aβ) in the brain. Aβ is derived from β-amyloid precursor protein (APP) through sequential cleavages by the β-secretase and the γ-secretase. In addition to Aβ, γ-cleavage releases the intracellular domain of APP (AICD). However, although it is well-established that Aβ is the prime culprit for AD pathogenesis, the physio/pathological functions of AICD remain largely elusive. Here we review recent progress toward elucidating the functional roles of AICD, which include modulating intracellular trafficking/processing of APP, inducing apoptosis, and regulating gene expression at transcriptional level.国家自然科学基金(30672198);; 福建省高等学校新世纪优秀人才支持计

Pathological and physiological functions of presenilins

Mutations in PSEN1 and PSEN2 genes account for the majority of cases of early-onset familial Alzheimer disease. Since the first prediction of a genetic link between PSEN1 and PSEN2 with Alzheimer's disease, many research groups from both academia and pharmaceutical industry have sought to unravel how pathogenic mutations in PSEN cause presenile dementia. PSEN genes encode polytopic membrane proteins termed presenilins (PS1 and PS2), which function as the catalytic subunit of γ-secretase, an intramembrane protease that has a wide spectrum of type I membrane protein substrates. Sequential cleavage of amyloid precursor protein by BACE and γ-secretase releases highly fibrillogenic β-amyloid peptides, which accumulate in the brains of aged individuals and patients with Alzheimer's disease. Familial Alzheimer's disease-associated presenilin variants are thought to exert their pathogenic function by selectively elevating the levels of highly amyloidogenic Aβ42 peptides. In addition to Alzheimer's disease, several recent studies have linked PSEN1 to familiar frontotemporal dementia. Here, we review the biology of PS1, its role in γ-secretase activity, and discuss recent developments in the cell biology of PS1 with respect to Alzheimer's disease pathogenesis

Genetic divergence of orangutan subspecies (Pongo pygmaeus)

Microsatellites and mitochondrial DNA sequences were studied for the two subspecies of orangutans (Pongo pygmaeus), which are located in Borneo (P. p, pygmaeus) and Sumatra (P. p. abelii), respectively. Both subspecies possess marked genetic diversity. Genetic subdivision was identified within the Sumatran orangutans. The genetic differentiation between the two subspecies is highly significant for ND5 region but not significant for 16s rRNA or microsatellite data by exact tests, although F-ST estimates are highly significant for these markers. Divergence time between the two subspecies is approximately 2.3 +/- 0.5 million years ago (MYA) estimated from our data, much earlier than the isolation of their geological distribution, Neither subspecies underwent a recent bottleneck, though the Sumatran subspecies might have experienced expansion approximately 82,000 years ago. The estimated effective population sizes for both subspecies are on the order of 10(4), Our results contribute additional information that may be interpreted in the context of orangutan conservation efforts

Differential regulation of BACE1 expression by oxidative and nitrosative signals

<p>Abstract</p> <p>Background</p> <p>It is well established that both cerebral hypoperfusion/stroke and type 2 diabetes are risk factors for Alzheimer's disease (AD). Recently, the molecular link between ischemia/hypoxia and amyloid precursor protein (APP) processing has begun to be established. However, the role of the key common denominator, namely nitric oxide (NO), in AD is largely unknown. In this study, we investigated redox regulation of BACE1, the rate-limiting enzyme responsible for the β-cleavage of APP to Aβ peptides.</p> <p>Results</p> <p>Herein, we studied events such as S-nitrosylation, a covalent modification of cysteine residues by NO, and H₂O₂-mediated oxidation. We found that NO and H₂O₂differentially modulate BACE1 expression and enzymatic activity: NO at low concentrations (<100 nM) suppresses BACE1 transcription as well as its enzymatic activity while at higher levels (0.1-100 μM) NO induces S-nitrosylation of BACE1 which inactivates the enzyme without altering its expression. Moreover, the suppressive effect on BACE1 transcription is mediated by the NO/cGMP-PKG signaling, likely through activated PGC-1α. H₂O₂(1-10 μM) induces BACE1 expression via transcriptional activation, resulting in increased enzymatic activity. The differential effects of NO and H₂O₂on BACE1 expression and activity are also reflected in their opposing effects on Aβ generation in cultured neurons in a dose-dependent manner. Furthermore, we found that BACE1 is highly S-nitrosylated in normal aging brains while S-nitrosylation is markedly reduced in AD brains.</p> <p>Conclusion</p> <p>This study demonstrates for the first time that BACE1 is highly modified by NO via multiple mechanisms: low and high levels of NO suppress BACE1 via transcriptional and post translational regulation, in contrast with the upregulation of BACE1 by H₂O₂-mediated oxidation. These novel NO-mediated regulatory mechanisms likely protect BACE1 from being further oxidized by excessive oxidative stress, as from H₂O₂and peroxynitrite which are known to upregulate BACE1 and activate the enzyme, resulting in excessive cleavage of APP and Aβ generation; they likely represent the crucial house-keeping mechanism for BACE1 expression/activation under physiological conditions.</p

Novel Pathways Regulating Function and Metabolism of ß-Amyloid Precursor Protein in Alzheimer's Disease

阿尔茨海默病(AD)是最常见的老年人痴呆病症，AD病人最显著的病理特征是细胞外的淀粉样斑和细胞内的神经纤维缠结(NFT)。淀粉样斑是由β-淀粉样蛋白(Aβ)组成，而NFT由细胞骨架蛋白tau组成。Aβ是由淀粉样前体蛋白(APP)经β-分泌酶和γ-分泌酶依次切割而成；而NFT的形成与tau蛋白异常磷酸化有关。多种证据表明，大脑中Aβ的过度产生和聚集是AD的主要原因：Aβ具有神经毒性并可触发级联反应引起细胞死亡。Alzheimer’s disease (AD) is the most common neurodegenera-tive disorder worldwide, defined by two classical hallmark pa-thologies: extracellular senile plaques and intraneuronal neu-rofibrillary tangles (NFTs) (1,2). NFTs are composed of the hyperphosphorylated microtubule-associated protein tau that is abnormally phosphorylated primarily by glycogen synthase ki-nase-3 (GSK-3) and cyclin D kinase 5 (Cdk5) (2). Senile plaques are composed of heterogeneous small peptides collectively called β-amyloid (Aβ), derived from the β-amyloid precursor pro-tein (APP) through sequential cleavage by β- and γ-secretases. APP is synthesized in the endoplasmic reticulum (ER) and trans-ported through the Golgi/trans-Golgi network (TGN) to the plasma membrane, where it can be cleaved by α-secretase to produce sAPPα. Non-cleaved APP is re-internalized and is subjected to amyloidogenic processing for Aβ generation (1). Multiple lines of evidence suggest that overproduction/aggregation of Aβ in the brain is the primary cause of AD: Aβ is highly toxic to neurons and can trigger a cascade of pathogenic events leading to cell death. Therefore, detailed delineation of the function, process-ing, and regulated trafficking of APP is crucial for understanding the mechanism underlying AD pathogenesis and for developing AD therapeutic strategies

Tumor suppressor PTEN affects tau phosphorylation: deficiency in the phosphatase activity of PTEN increases aggregation of an FTDP-17 mutant Tau

BACKGROUND: Aberrant hyperphosphorylation of tau protein has been implicated in a variety of neurodegenerative disorders. Although a number of protein kinases have been shown to phosphorylate tau in vitro and in vivo, the molecular mechanisms by which tau phosphorylation is regulated pathophysiologically are largely unknown. Recently, a growing body of evidence suggests a link between tau phosphorylation and PI3K signaling. In this study, phosphorylation, aggregation and binding to the microtubule of a mutant frontal temporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) tau in the presence of tumor suppressor PTEN, a major regulatory component in PI3K signaling, were investigated. RESULTS: Phosphorylation of the human mutant FTDP-17 tau, T40RW, was evaluated using different phospho-tau specific antibodies in the presence of human wild-type or phosphatase activity null mutant PTEN. Among the evaluated phosphorylation sites, the levels of Ser214 and Thr212 phospho-tau proteins were significantly decreased in the presence of wild-type PTEN, and significantly increased when the phosphatase activity null mutant PTEN was ectopically expressed. Fractionation of the mutant tau transfected cells revealed a significantly increased level of soluble tau in cytosol when wild-type PTEN was expressed, and an elevated level of SDS-soluble tau aggregates in the presence of the mutant PTEN. In addition, the filter/trap assays detected more SDS-insoluble mutant tau aggregates in the cells overexpressing the mutant PTEN compared to those in the cells overexpressing wild-type PTEN and control DNA. This notion was confirmed by the immunocytochemical experiment which demonstrated that the overexpression of the phosphatase activity null mutant PTEN caused the mutant tau to form aggregates in the COS-7 cells. CONCLUSION: Tumor suppressor PTEN can alleviate the phosporylation of the mutant FTDP-17 tau at specific sites, and the phosphatase activity null PTEN increases the mutant tau phosphorylation at these sites. The changes of the tau phosphorylation status by ectopic expression of PTEN correlate to the alteration of the mutant tau's cellular distribution. In addition, the overexpression of the mutant PTEN can increase the level of the mutant tau aggregates and lead to the formation of visible aggregates in the cells

一种中间格网可组合调换式水生植物共培养装置

本实用新型公开了一种中间格网可组合调换式水生植物共培养装置，玻璃凸条用玻璃胶固定在矩形共培养玻璃缸的第一长侧面、第二长侧面的中间位置，共培养框架两侧的凹槽尺寸与玻璃凸条大小相匹配，共培养格网可根据实验需要选择单面或双面覆于共培养格网框架的正反两个框面，四个角落均用铆钉插于共培养框架的四个铆钉孔中来固定格网，固定好共培养格网的共培养框架对准玻璃凸条插入。该共培养装置结构简单，操作方便，成本低廉，比传统共培养玻璃缸具有更高利用率和研究效果。实验结束后的格网能方便的从框架上取下，清洗、晾干、消毒后能重复利用，大幅度减小了实验误差，提高了实验效率以及实验的准确率、精确度。</p

Transcriptional regulation of PEN-2, a key component of the γ-secretase complex, by CREB

Gamma-secretase, which is responsible for the intramembranous cleavage of Alzheimer's P-amyloid precursor protein (APP), the signaling receptor Notch, and many other substrates, is a multiprotein complex consisting of at least four components: presenilin (PS), nicastrin, APH-1, and PEN-2. Despite the fact that PEN-2 is known to mediate endoproteolytic cleavage of full-length PS and APH-1 and nicastrin are required for maintaining the stability of the complex, the detailed physiological function of each component remain elusive. Unlike that of PS, the transcriptional regulation of PEN-2, APH-1, and nicastrin has not been investigated. Here, we characterized the upstream regions of the human PEN-2 gene and identified a 238-bp fragment located 353 bp upstream of the translational start codon as the key region necessary for the promoter activity. Further analysis revealed a CREB binding site located in the 238-bp region that is essential for the transcriptional activity of the PEN-2 promoter. Mutation of the CREB site abolished the transcriptional activity of the PEN-2 promoter. Electrophoretic mobility shift assays and chromatin immunoprecipitation analysis showed the binding of CREB to the PEN-2 promoter region both in vitro and in vivo. Activation of the CREB transcriptional factor by forskolin dramatically promoted the expression of PEN-2 mRNA and protein, whereas the other components of the gamma-secretase complex remained unaffected. Forskolin treatment slightly increases the secretion of soluble APP alpha and A beta without affecting Notch cleavage. These results demonstrate that expression of PEN-2 is regulated by CREB and suggest that the specific control of PEN-2 expression may imply additional physiological functions uniquely assigned to PEN-2

Sorting nexin 12 interacts with BACE1 and regulates BACE1-mediated APP processing

Background: beta-site APP cleaving enzyme 1 (BACE1) cleaves beta-amyloid precursor protein (APP) to initiate the production of beta-amyloid (A beta), the prime culprit in Alzheimer's disease (AD). Dysregulation of the intracellular trafficking of BACE1 may affect A beta generation, contributing to AD pathology. In this study, we investigated whether BACE1 trafficking and BACE1-mediated APP processing/A beta generation are affected by sorting nexin 12 (SNX12), a member of the sorting nexin (SNX) family that is involved in protein trafficking regulation. Results: Herein, we find that SNX12 is widely expressed in brain tissues and is mainly localized in the early endosomes. Overexpression of SNX12 does not affect the steady-state levels of APP, BACE1 or gamma-secretase components, but dramatically reduces the levels of A beta, soluble APP beta and APP beta-carboxyl terminal fragments. Downregulation of SNX12 has the opposite effects. Modulation of SNX12 levels does not affect gamma-secretase activity or in vitro beta-secretase activity. Further studies reveal that SNX12 interacts with BACE1 and downregulation of SNX12 accelerates BACE1 endocytosis and decreases steady-state level of cell surface BACE1. Finally, we find that the SNX12 protein level is dramatically decreased in the brain of AD patients as compared to that of controls. Conclusion: This study demonstrates that SNX12 can regulate the endocytosis of BACE1 through their interaction, thereby affecting beta-processing of APP for A beta production. The reduced level of SNX12 in AD brains suggests that an alteration of SNX12 may contribute to AD pathology. Therefore, inhibition of BACE1-mediated beta-processing of APP by regulating SNX12 might serve as an alternative strategy in developing an AD intervention.Alzheimer's Association; National Natural Science Foundation of China [30973150, 81161120496, 81000540]; 973 Prophase Project [2010CB535004]; Natural Science Foundation of Fujian Province of China [2009J06022, 2010J01235]; Program for New Century Excellent Talents in Universities (NCET); Fundamental Research Funds for the Central Universities; Fok Ying Tung Education Foundatio

p53-dependent control of transactivation of the Pen2 promoter by presenilins

The senile plaques found in the brains of patients with Alzheimer's disease are mainly due to the accumulation of amyloid β-peptides (Aβ) that are liberated by γ-secretase, a high molecular weight complex including presenilins, PEN-2, APH-1 and nicastrin. The depletion of each of these proteins disrupts the complex assembly into a functional protease. Here, we describe another level of regulation of this multimeric protease. The depletion of both presenilins drastically reduces Pen2 mRNA levels and its promoter transactivation. Furthermore, overexpression of presenilin-1 lowers Pen2 promoter transactivation, a phenotype abolished by a double mutation known to prevent presenilin-dependent γ-secretase activity. PEN-2 expression is decreased by depletion of β-amyloid precursor protein (APP) and increased by the APP intracellular domain (AICD). We show that AICD and APP complement for Pen2 mRNA levels in APP/APLP1-2 knockout fibroblasts. Interestingly, overexpression of presenilin-2 greatly increases Pen2 promoter transactivation. The opposite effect triggered by both presenilins was reminiscent of our previous study, which showed that these two proteins elicit antagonistic effects on p53. Therefore, we examined the contribution of p53 on Pen2 transcription. Pen2 promoter transactivation, and Pen2 mRNA and protein levels were drastically reduced in p53–/– fibroblasts. Furthermore, PEN-2 expression could be rescued by p53 complementation in p53- and APP-deficient cells. Interestingly, PEN-2 expression was also reduced in p53-deficient mouse brain. Overall, our study describes a p53-dependent regulation of PEN-2 expression by other members of the γ-secretase complex, namely presenilins