Ac-HF对Aβ1-42致大鼠皮质原代神经元损伤的保护作用及对α分泌酶活性的影响

目的 探讨贯叶金丝桃素乙酸酯(hyperforin acetate,Ac-HF)对β淀粉样肽(Aβ)1-42毒性损伤大鼠原代培养大脑皮层神经元的作用及对α分泌酶活性的影响及可能机制.方法 采用大鼠原代培养皮层细胞,用MTT法观察Ac-HF对Aβ1-42毒性损伤后的神经细胞活力的影响.用ELASA检测其对α分泌酶活性及可溶性淀粉样前体蛋白(sAPPα)分泌的影响,应用Western blot检其对淀粉样前体蛋白(APP)及解聚金属蛋白酶(a disintegrin and metalloproteinas,ADAM)10表达的影响;并观察Calphostin C(Cal.C)对Ac-HF这些作用的影响.结果 0.1～10.0 μmol/L Ac-HF无细胞毒性(P＞0.05),1.0～50.0 μmol/L Ac-HF可增加Aβ1-42毒性损伤的大鼠皮层神经细胞的活力(P＜0.05),0.1～10.0 μmol/L Ac-HF可使α分泌酶活性增加,sAPPα分泌增多,对APP蛋白表达无影响,使ADAM10表达增多,PKC抑制剂Calphostin C(Cal.C)可抑制Ac-HF对α分泌酶的活性,sAPPα分泌及ADAM10表达的影响.结论 1.0～10.0 μmol/L Ac-HF对Aβ1-42毒性损伤的大鼠皮层神经细胞有保护作用,Ac-HF可通过增加α分泌酶活性及sAPPα分泌产生保护作用,这一作用可能是通过激活PKC通路增加ADAM10表达.     

Perlecan domain V inhibits α2 integrin-mediated amyloid-β neurotoxicity

Amyloid-β (Aβ) peptide is a key component of amyloid plaques, one of the pathological features of Alzheimer's disease. Another feature is pronounced cell loss in the brain leading to an enlargement of the ventricular area and a decrease in brain weight and volume. Aβ plaque deposition and neuronal toxicity can be modeled by treating human cortical neuronal cultures with Aβ and showing robust Aβ deposition and neurotoxicity that is mediated by α2β1 and αvβ1 integrins. The current study expands on these findings by showing that the domain V of perlecan, a known α2 integrin ligand, inhibits Aβ neurotoxicity in an α2 integrin-dependent manner. Additionally, Aβ binds more efficiently to cells expressing activated α2 integrin. Finally the inhibition of Aβ neurotoxicity with domain V is synergistic with inhibitors of αv integrin and β1 integrin. We propose that domain V and potentially other α2 integrin ligands could be a new therapeutic approach for inhibiting the Aβ plaque deposition and neurotoxicity observed in Alzheimer's disease.     

Cryptotanshinone,a compound from Salvia miltiorrhiza modulates amyloid precursor protein metabolism and attenuates β-amyloid deposition through upregulating α-secretase in vivo and in vitro

The amyloid precursor protein (APP) is cleaved enzymatically by non-amyloidogenic and amyloidogenic pathways. α-Secretase cleaves APP within β-amyloid protein (Aβ) sequence, resulting in the release of a secreted fragment of APP (sAPPα) and precluding Aβ generation. Cryptotanshinone (CTS), an active component of the medicinal herb Salvia miltiorrhiza, has been shown to improve learning and memory in several pharmacological models of Alzheimer's disease (AD). However, the effects of CTS on the Aβ plaque pathology and the APP processing in AD are unclear. Here we reported that CTS strongly attenuated amyloid plaque deposition in the brain of APP/PS1 transgenic mice. In addition, CTS significantly improved spatial learning and memory in APP/PS1 mice assessed by the Morris water maze testing. To define the exact molecular mechanisms involved in the beneficial effects of CTS, we investigated the effects of the CTS on APP processing in rat cortical neuronal cells overexpressing Swedish mutant human APP695. CTS was found to decrease Aβ generation in concentration-dependent (0–10 μM) manner. Interestingly, the N-terminal APP cleavage product, sAPPα was markedly increased by CTS. Further study showed that α-secretase activity was increased by CTS. Taken together, our results suggested CTS improved the cognitive ability in AD transgenic mice and promoted APP metabolism toward the non-amyloidogenic products pathway in rat cortical neuronal cells. CTS shows a promising novel way for the therapy of AD.     

Salidroside attenuates hypoxia-induced abnormal processing of amyloid precursor protein by decreasing BACE1 expression in SH-SY5Y cells

Hypoxia which is mainly mediated by hypoxia-inducible factor 1 (HIF-1), can greatly contribute to the occurrence of Alzheimer's disease (AD) by increasing β-site APP cleaving enzyme (BACE1) gene expression, protein level and β-secretase activity, resulting in a significant generation of amyloid-beta (Aβ). Salidroside has been reported to have great neuroprotective effects. The aim of this study was to investigate the effects of salidroside on hypoxia-induced abnormal processing of the amyloid precursor protein (APP) in SH-SY5Y cells and its possible mechanism. Western blot analysis showed that 200 μM of salidroside pretreatment significantly decreased BACE1 protein level and promoted the secretion of sAPPα in hypoxic condition. Salidroside had no effect on the level of APP, ADAM10 and ADAM17. ELISA analysis revealed that salidroside was able to inhibit the increase of β-secretase activity and Aβ generation induced by hypoxia, with no effect on γ-secretase activity. Notably, under hypoxia condition, mRNA of BACE1 and protein level of HIF-1α were decreased by salidroside pretreatment. These results demonstrated for the first time that salidroside was able to attenuate abnormal processing of amyloid precursor protein induced by hypoxia in SH-SY5Y cells, providing a new insight into prevention and treatment of Alzheimer's disease.     

Chronic 5-HT4 receptor activation decreases Aβ production and deposition in hAPP/PS1 mice

Lowering the production and accumulation of Aβ has been explored as treatment for Alzheimer's disease (AD), because Aβ is postulated to play an important role in the pathogenesis of AD. 5-HT₄ receptors are an interesting drug target in this regard, as their activation might stimulate α-secretase processing, which increases sAPPα and reduces Aβ, at least according to the central dogma in APP processing. Here we describe a novel high-affinity 5-HT₄ receptor agonist SSP-002392 that, in cultured human neuroblastoma cells, potently increases the levels of cAMP and sAPPα at 100-fold lower concentrations than the effective concentrations of prucalopride, a known selective 5-HT₄ receptor agonist. Chronic administration of this compound in a hAPP/PS1 mouse model of Alzheimer's disease decreased soluble and insoluble Aβ in hippocampus, but the potential mechanisms underlying these observations seem to be complex. We found no evidence for direct α-secretase stimulation in the brain in vivo, but observed decreased APP and BACE-1 expression and elevated astroglia and microglia responses. Taken together these results provide support for a potential disease-modifying aspect when stimulating central 5-HT₄ receptors; however, the complexity of the phenomena warrants further research.     

Nardilysin prevents amyloid plaque formation by enhancing α-secretase activity in an Alzheimer's disease mouse model

Amyloid beta (Aβ) peptide, the main component of senile plaques in patients with Alzheimer's disease (AD), is derived from proteolytic cleavage of amyloid precursor protein (APP) by β- and γ-secretases. Alpha-cleavage of APP by α-secretase has a potential to preclude the generation of Aβ because it occurs within the Aβ domain. We previously reported that a metalloendopeptidase, nardilysin (N-arginine dibasic convertase; NRDc) enhances α-cleavage of APP, which results in the decreased generation of Aβ in vitro. To clarify the in vivo role of NRDc in AD, we intercrossed transgenic mice expressing NRDc in the forebrain with an AD mouse model. Here we demonstrate that the neuron-specific overexpression of NRDc prevents Aβ deposition in the AD mouse model. The activity of α-secretase in the mouse brain was enhanced by the overexpression of NRDc, and was reduced by the deletion of NRDc. However, reactive gliosis adjacent to the Aβ plaques, one of the pathological features of AD, was not affected by the overexpression of NRDc. Taken together, our results indicate that NRDc controls Aβ formation through the regulation of α-secretase.     

In vivo P2X7 inhibition reduces amyloid plaques in Alzheimer's disease through GSK3β and secretases

β-Amyloid (Aβ) peptide production from amyloid precursor protein (APP) is essential in the formation of the β-amyloid plaques characteristic of Alzheimer's disease. However, the extracellular signals that maintain the balance between nonpathogenic and pathologic forms of APP processing, mediated by α-secretase and β-secretase respectively, remain poorly understood. In the present work, we describe regulation of the processing of APP via the adenosine triphosphate (ATP) receptor P2X7R. In 2 different cellular lines, the inhibition of either native or overexpressed P2X7R increased α-secretase activity through inhibition of glycogen synthase kinase 3 (GSK-3). In vivo inhibition of the P2X7R in J20 mice, transgenic for mutant human APP, induced a significant decrease in the number of hippocampal amyloid plaques. This reduction correlated with a decrease in glycogen synthase kinase 3 activity in J20 mice, increasing the proteolytic processing of APP through an increase in α-secretase activity. The in vivo findings presented here demonstrate for the first time the therapeutic potential of P2X7R antagonism in the treatment of familiar Alzheimer's disease (FAD).     

Cytoprotective response induced by electromagnetic stimulation on SH-SY5Y human neuroblastoma cell line

It is well known that physiological functions and pathological conditions of cells and tissues can be influenced not only by chemical molecules, but also by physical stimuli such as electromagnetic waves. In particular, epidemiological studies suggest possible associations between exposure to electromagnetic fields and an increased risk of tumors and neurodegenerative disorders, such as Alzheimer's disease. However, depending on the dose and on the length of treatment, the electromagnetic stimuli can be harmful or induce a cytoprotective cellular response, suggesting a possible application in medical therapy. In this study, under a tissue engineering viewpoint, we investigated the effects of an electromagnetic wave (magnetic field intensity, 2 mT; frequency, 75 Hz) on a neuronal cellular model characterized by the overexpression of the amyloid precursor protein (APP). After a prolonged electromagnetic treatment, lower mitochondrial activity and proliferation rate, resulting in a higher cellular quiescence, were observed. Focusing on the stress and oxidative pathways, we detected an overall increase of two fundamental proteins, the chaperone heat shock protein HSP70 and the free radical scavenger superoxide dismutase-1 enzyme (SOD-1). Interestingly, we found that the electromagnetic stimulation promotes the nonamyloidogenic processing of APP through an increased expression of the α-secretase ADAM10 and an enhanced release of the soluble neurotrophic factor sAPPα (a product of the ADAM10-mediated cleavage of APP). In conclusion, these findings suggest that the electromagnetic stimulus, if properly administered in terms of dose and timing, is able to induce a cytoprotective response in the cell. Moreover, these results suggest a possible use of this particular physical stimulation to improve the functional capability of the cells to face noxae.     

Selective neutralization of APP-C99 with monoclonal antibodies reduces the production of Alzheimer's Aβ peptides

The toxic amyloid-β (Aβ) peptides involved in Alzheimer's disease (AD) are produced after processing of the amyloid precursor protein-C-terminal fragment APP-C99 by γ-secretase. Thus, major therapeutic efforts have been focused on inhibiting the activity of this enzyme. However, preclinical and clinical trials testing γ-secretase inhibitors revealed adverse side effects most likely attributed to impaired processing of the Notch-1 receptor, a γ-secretase substrate critically involved in cell fate decisions. Here we report an innovative approach to selectively target the γ-secretase-mediated processing of APP-C99 with monoclonal antibodies neutralizing this substrate. Generated by immunizing mice with natively folded APP-C99, these antibodies bound N- or C-terminal accessible epitopes of this substrate, and decorated extracellular amyloid deposits in AD brain tissues. In cell-based assays, the same antibodies impaired APP-C99 processing by γ-secretase, and reduced Aβ production. Furthermore, they significantly decreased brain Aβ levels in the APPPS1 mouse model of AD after intracerebroventricular injection. Together, our findings support APP-C99 substrate-targeting antibodies as new immunotherapeutic and Notch-sparing agents to lower the levels of Aβ peptides implicated in AD.     

Pharmacologic blockade of 5-lipoxygenase improves the amyloidotic phenotype of an Alzheimer's disease transgenic mouse model involvement of γ-secretase

The 5-lipoxygenase (5-LO) enzyme is widely distributed within the central nervous system. Previous works showed that this protein is up-regulated in Alzheimer's disease (AD) and that its genetic absence results in a reduction of amyloid β (Aβ) levels in Tg2576 mice. In the present study, we examined the effect of 5-LO pharmacological inhibition on the amyloidotic phenotype of these mice. Aβ deposition in the brains of mice receiving zileuton, a selective and specific 5-LO inhibitor, was significantly reduced when compared with control Tg2576 mice receiving vehicle. This reduction was associated with a similar decrease in brain Aβ peptides levels. Zileuton treatment did not induce any change in the steady state levels of amyloid-β precursor protein (APP), BACE1 or ADAM10. By contrast, it resulted in a significant reduction of presenilin 1 (PSEN1, alias PS1), nicastrin (NCSTN) , presenilin enhancer 2 homolog (PSNEN, alias, Pen-2), and anterior pharynx defective 1 (APH-1), the four components of the γ-secretase complex-at the protein and message level. Furthermore, in vitro studies confirmed that zileuton prevents Aβ formation by modulating γ-secretase complex levels without affecting Notch signaling. These data establish a functional role for 5-LO in the pathogenesis of AD-like amyloidosis, whereby it modulates the γ-secretase pathway. They suggest that pharmacological inhibition of 5-LO could provide a novel therapeutic opportunity for AD.     

锌转运体ZNT1在人阿尔茨海默病大脑的定位

目的研究锌转运体-1(zinc transporter 1,ZNT1)在阿尔茨海默病(Alzheimer's Disease,AD)尸检大脑皮层内的定位分布,探讨ZNT1影响脑锌平衡从而参与AD发病的可能机制。方法应用免疫组织化学、免疫荧光和共聚焦激光扫描显微镜观察ZNT1蛋白和β-淀粉样蛋白(β-amyloid,Aβ)在AD患者大脑内的分布和二者之间的位置关系。结果ZNT1免疫阳性反应产物主要定位于AD大脑皮层老年斑内。几乎所有的Aβ老年斑内均有不同程度ZNT1表达。ZNT1和β-淀粉样蛋白免疫双标结果进一步证实ZNT1免疫产物与Aβ共存于AD患者的老年斑内。结论AD患者大脑皮质Aβ老年斑内有大量ZNT1蛋白表达,提示ZNT1可能参与AD大脑皮质Aβ老年斑的形成。     

细胞因子白细胞介素-1α、S100β 在阿尔茨海默病不同类型老年斑中的表达

目的 探讨小胶质细胞、星形胶质细胞及其所产生的炎性介质白细胞介素(IL)-1α、S100β在阿尔茨海默病（Alzheimer's disease,AD）不同类型的老年斑形成和进展中的意义。方法 从北京医院病理科1982至2008年尸检资料中选出AD 34例,所有脑标本的海马部切片均进行IL-1α/β-淀粉蛋白、S100β/β-淀粉蛋白免疫组织化学双标记染色。结果 IL-1α/β-淀粉蛋白及S100 β/ β-淀粉蛋白免疫组织化学双标记染色均显示老年斑有4种类型,即弥漫性非神经突斑、弥漫性神经突斑、有致密核心的神经突斑、有致密核心的非神经突斑。在4种类型的老年斑中,与弥漫性神经突斑相关的小胶质细胞和星形胶质细胞数量最多,分别为(7.29±3.04)和(6.49±2.20)个/ mm2,与弥漫性非神经突斑、有致密核心的神经突斑及有致密核心的非神经突斑的小胶质细胞和星形胶质细胞的数量,分别为(3.24±1.53)和(4.14±1.77)个/mm2,(2.09±1.37)和(2.25±0.83)个/ mm2,(1.38±0.90)和(0.58±0.36)个/mm2,与弥漫性神经突斑相关的小胶质细胞和星形胶质细胞数量均高于其他类型的老年斑(P＜0.05)。结论 小胶质细胞、星形胶质细胞及其所产生的炎性介质IL-1α、S100β可能在AD弥漫性非神经突斑转化为弥漫性神经突斑的过程中具有一定意义。     

Neuronal activity regulates the regional vulnerability to amyloid-β deposition

Amyloid-β (Aβ) plaque deposition in specific brain regions is a pathological hallmark of Alzheimer's disease. However, the mechanism underlying the regional vulnerability to Aβ deposition in Alzheimer's disease is unknown. Herein, we provide evidence that endogenous neuronal activity regulates the regional concentration of interstitial fluid (ISF) Aβ, which drives local Aβ aggregation. Using in vivo microdialysis, we show that ISF Aβ concentrations in several brain regions of APP transgenic mice before plaque deposition were commensurate with the degree of subsequent plaque deposition and with the concentration of lactate, a marker of neuronal activity. Furthermore, unilateral vibrissal stimulation increased ISF Aβ, and unilateral vibrissal deprivation decreased ISF Aβ and lactate, in contralateral barrel cortex. Long-term unilateral vibrissal deprivation decreased amyloid plaque formation and growth. Our results suggest a mechanism to account for the vulnerability of specific brain regions to Aβ deposition in Alzheimer's disease.     

Memantine treatment decreases levels of secreted Alzheimer's amyloid precursor protein (APP) and amyloid beta (Aβ) peptide in the human neuroblastoma cells

Memantine, an uncompetitive NMDA receptor antagonist, is a FDA-approved drug used for the treatment of moderate-to-severe Alzheimer's disease (AD). Several studies have documented protective roles of memantine against amyloid beta (Aβ) peptide-mediated damage to neurons in both in vitro and in vivo models. Memantine is also effective in reducing amyloid burden in the brain of APP transgenic mice. However, the exact mechanism by which memantine provides protection against Aβ-mediated neurodegenerative cascade, including APP metabolism, remains to be elucidated. Herein, we investigated the effect of memantine on levels of the secreted form of Aβ precursor protein (APP), secreted Aβ and cell viability markers under short/acute conditions. We treated neuronal SK-N-SH cells with 10 μM memantine and measured levels of secreted total APP (sAPP), APPα isoform and Aβ_(1–40) in a time dependent manner for up to 24 h. Memantine significantly decreased the levels of the secreted form of sAPP, sAPPα and Aβ_(1–40) compared to vehicle treated cells. This change started as early as 8 h and continued for up to 24 h of drug treatment. Unlike sAPP, a slight non-significant increase in total intracellular APP level was observed in 24-h treated memantine cells. Taken together, these results suggest a role for memantine in the transport or trafficking of APP molecules away from the site of their proteolytic cleavage by the secretase enzymes. Such a novel property of memantine warrants further study to define its therapeutic utility.     

Physiological functions of the amyloid precursor protein secretases ADAM10, BACE1, and Presenilin

Alzheimer's disease causing mutations in the amyloid precursor protein (APP) or in the Presenilin 1 (PS1) or Presenilin 2 (PS2) genes increase the production of amyloid peptides (Aβ) that precipitate in amyloid plaques. Since amyloid plaques are also a prominent feature of sporadic Alzheimer's disease (AD), abnormal proteolysis of APP and the generation of amyloid beta (Aβ) are key events in the pathogenesis of AD. The proteases (secretases) that cleave APP are therefore important therapeutic targets, both for the rare familial forms but likely also for the sporadic forms of AD. The identification and understanding of the (neuro)biological functions of the α-, β-, and presenilin/γ-secretase (complexes) is important for the development of drugs and the delineation of their associated side effects. The potential impact of this type of research exceeds the AD field since the function of these secretases are also linked to cellular pathways like ectodomain shedding of growth factors and regulated intramembrane proteolysis of receptors in developmental biology, tissue homeostasis, and tumorigenesis. The generation of mice deficient in presenilin 1, presenilin 2, the α-secretase ADAM10, and the β-secretases BACE1 and BACE2 were instrumental for the elucidation of the physiological functions of these proteases. Using these mouse models understanding how these secretases regulate amyloid peptide formation and how they exert their diverse biological functions could be significantly increased. This review attempts to summarize selected aspects of the current view of the multiple roles such proteases play in health and disease.     

Pro-oxidant diet enhances β/γ secretase-mediated APP processing in APP/PS1 transgenic mice

The etiology of Alzheimer's disease (AD) is complex with oxidative stress being a possible contributory factor to pathogenesis and disease progression. TASTPM transgenic mice expressing familial AD-associated amyloid precursor protein (APPswe) and presenilin transgenes (PS1M146V) show increased brain amyloid beta (Aβ) levels and Aβ plaques from 3 months. We tested if enhancing oxidative stress through diet would accelerate Aβ-related pathology. TASTPM were fed a pro-oxidant diet for 3 months resulting in increased brain levels of protein carbonyls, increased Nrf2, and elevated concentrations of glutathione (GSH). The diet increased both amyloid precursor protein (APP) and Aβ in the cortex of TASTPM but did not alter Aβ plaque load, presenilin 1, or β-secretase (BACE1) expression. TASTPM cortical neurons were cultured under similar pro-oxidant conditions resulting in increased levels of APP and Aβ likely as a result of enhanced β/γ secretase processing of APP. Thus, pro-oxidant conditions increase APP levels and enhance BACE1-mediated APP processing and in doing so might contribute to pathogenesis in AD.     

Amyloid precursor protein (APP) processing genes and cerebrospinal fluid APP cleavage product levels in Alzheimer's disease

The aim of this exploratory investigation was to determine if genetic variation within amyloid precursor protein (APP) or its processing enzymes correlates with APP cleavage product levels: APPα, APPβ or Aβ42, in cerebrospinal fluid (CSF) of cognitively normal subjects or Alzheimer's disease (AD) patients. Cognitively normal control subjects (n = 170) and AD patients (n = 92) were genotyped for 19 putative regulatory tagging SNPs within 9 genes (APP, ADAM10, BACE1, BACE2, PSEN1, PSEN2, PEN2, NCSTN and APH1B) involved in the APP processing pathway. SNP genotypes were tested for their association with CSF APPα, APPβ, and Aβ42, AD risk and age-at-onset while taking into account age, gender, race and APOE ε4. After adjusting for multiple comparisons, a significant association was found between ADAM10 SNP rs514049 and APPα levels. In controls, the rs514049 CC genotype had higher APPα levels than the CA, AA collapsed genotype, whereas the opposite effect was seen in AD patients. These results suggest that genetic variation within ADAM10, an APP processing gene, influences CSF APPα levels in an AD specific manner.     

Angiotensin II does not directly affect Aβ secretion or β-/γ-secretase activity via activation of angiotensin II type 1 receptor

Recent studies suggest that brain angiotensin II (Ang II), the major effector molecule of the renin-angiotensin system, is implicated in the pathogenesis of Alzheimer's disease (AD). However, it remains unknown whether activation or blockade of the angiotensin II type 1 receptor (AT1R) has an impact on the secretion of amyloid-β (Aβ), the key molecule in the pathogenesis of AD. In this study, the cell models cultured primary hippocampal neurons and human embryonic kidney 293 cells, were transfected with AT1R and amyloid precursor protein/presenilin-1 (PS1). The effects of activation/blockade of the AT1R on Aβ secretion, PS1 level and β-/γ-secretase activity were investigated in different cells. When AT1R was stimulated with Ang II at concentrations from 10nM to 1000nM, only a tendency toward increased Aβ secretion and β-/γ-secretase activity was noticed in cultured primary hippocampal neurons. However, no significant change in soluble Aβ(40) or Aβ(42), the level of PS1, or secretase activity was found in the cells. Similarly, when the AT1R was blocked by losartan, no significant alteration of Aβ secretion, PS1 levels or secretase activity was detected. In conclusion, this in vitro study demonstrates that Ang II does not directly affect Aβ secretion or secretase activity via activation of AT1R. This study is significantly meaningful for exploring the pharmacologic mechanisms of angiotensin II receptor blockers in AD.