Identification of Heparin-Binding Domains in the Amyloid Precursor Protein of Alzheimer's Disease by Deletion Mutagenesis and Peptide Mapping

Abstract: Recent studies have shown that the binding of the amyloid protein precursor (APP) of Alzheimer's disease to heparan sulfate proteoglycans (HSPGs) can modulate a neurite outgrowth-promoting function associated with APP. We used three different approaches to identify heparin-binding domains in APP. First, as heparin-binding domains are likely to be within highly folded regions of proteins, we analyzed the secondary structure of APP using several predictive algorithms. This analysis showed that two regions of APP₆₉₅ contain a high degree of secondary structure, and clusters of basic residues, considered mandatory for heparin binding, were found principally within these regions. To determine which domains of APP bind heparin, deletion mutants of APP₆₉₅ were prepared and analyzed for binding to a heparin affinity column. The results suggested that there must be at least two distinct heparin-binding regions in APP. To identify novel heparin-binding regions, peptides homologous to candidate heparin-binding domains were analyzed for their ability to bind heparin. These experiments suggested that APP contains at least four heparin-binding domains. The presence of more than one heparin-binding domain on APP suggests the possibility that APP may interact with more than one type of glycosaminoglycan.     

Vasopressin and Bradykinin Regulate Secretory Processing of the Amyloid Protein Precursor of Alzheimer's Disease

The amyloid protein precursor (APP) can be processed via several alternative processing pathways, -secretase processing by cleavage within the amyloid -peptide domain of APP is highly regulated by several external and internal signals including G protein-coupled receptors, protein kinase C and phospholipase A₂. In order to demonstrate that G protein-coupled neuropeptide receptors for bradykinin and vasopressin can increase -secretase processing of APP, we stimulated endogenously expressed bradykinin or vasopressin receptors in cell culture with the neuropeptides and measured the secreted ectodomain (APPs) in the conditioned media. Both bradykinin and vasopressin rapidly increased phosphatidylinositol (PI) turnover in PC-12 and in NRK-49F cells, indicating that these cell lines constitutively expressed functional PI-linked receptors for these neuropeptides. Both bradykinin and vasopressin readily stimulated APPs secretion. Increased APPs secretion was concentration-dependent and saturable, and it was blocked by receptor antagonists indicating specific receptor interaction of the peptides. The bradykinin-induced increase in APPs secretion in PC-12 cells was mediated by protein kinase C (PKC), whereas vasopressin receptors in NRK-49F cells were coupled to APP processing by PKC-independent signalling pathways. Our data show that neuropeptides can modulate APP processing in cell culture. In as much as increased -secretase processing is associated with decreased formation of A_1–40, a major constituent of amyloid plaques, our findings suggest a possible role for modulating neuropeptide receptors as a strategy for altering amyloid metabolism in Alzheimer's disease brain.     

Expression of the Chondroitin Sulfate Proteoglycans of Amyloid Precursor (Appican) and Amyloid Precursor-Like Protein 2

Abstract: The Alzheimer amyloid precursor (APP) protein is a member of a family of glycoproteins that includes the amyloid precursor-like proteins (APLPs). Previously, we showed that in C6 glioma cell cultures, secreted APP nexin II occurs as the core protein of a chondroitin sulfate proteoglycan (CSPG). Here, we report that among seven untransfected cell lines, expression of secreted APP CSPG was restricted to two cell lines of neural origin, namely, C6 glioma and Neuro-2a neuroblastoma (N2a) cells. Addition of dibutyryl cyclic AMP in N2a cultures, a treatment that induces the neuronal phenotype in these cells, resulted in a significant reduction in the amount of the secreted APP CSPG, although secretion of APP was only marginally affected. Growth in the presence of serum increased the size of the secreted APP CSPG, suggesting that the number and/or length of the chondroitin sulfate (CS) chains attached to the core APP varies with growth conditions. Extensive mapping with epitope-specific anti-bodies suggested that a CS chain is attached within or proximal to the Aβ sequence of APP. In contrast to the restricted expression of the APP CSPG, expression of secreted APLP2 CSPGs was observed in all cell lines examined. After chondroitinase treatment, two core proteins of ∼100 and 110 kDa were obtained that reacted with an APLP2-specific antiserum, suggesting that non-transfected cell lines contain at least two endogenous APLP2 CSPGs, probably derived by alternative splicing of the APLP2 KPI domain. The fraction of the APLP2 proteins in the CSPG form was dependent on the particular cell line examined. The proteoglycan nature of APP and APLP2 suggests that addition of the CS glycosaminoglycan chains is important for the implementation of the biological function of these proteins. However, the differential expression of these two proteoglycans suggests that their physiological roles and their possible involvement in Alzheimer's disease may differ.     

Expression of the Amyloid Precursor Protein of Alzheimer's Disease on the Surface of Transfected HeLa Cells

The principal component of the amyloid which accumulates in Alzheimer's Disease brain is a 4-kDa βA4 fragment of the amyloid precursor protein (APP). Although APP has the structural features of an integral transmembrane receptor, there has been limited evidence for expression of APP at the plasma membrane. The function of APP and related molecules is unknown. Using rabbit antisera to purified human brain APP, surface labeling of APP is demonstrable in HeLa cells transfected with the APP₆₉₅ isoform. Indirect immunofluorescence indicates the presence of APP at the surface of unfixed or aldehyde-fixed cells; preembedding immunoelectron microscopy using 5- or 1-nm gold particles and silver enhancement confirms plasma membrane labeling as well as labeling within intracellular membrane vesicles. Immunolabeling of unfixed cells at 4°C followed by incubation at 37°C shows APP within endocytic vesicles. Transfected HeLa cells with prominent surface APP were larger with more extensive microvilli than nonimmunoreactive HeLa cells. This is consistent with the postulated role of APP as a mediator of cell surface adhesion and membranematrix stabilization.     

Secretogranin I/Chromogranin B Is a Heparin-Binding Adhesive Protein

The major heparin-binding protein secreted by PC12 cells was purified from conditioned medium. Amino-terminal sequencing of the purified protein identified it as secretogranin I/chromogranin B (SgI/ChmB). The protein showed the same electrophoretic mobility and biochemical characteristics as previously reported for SgI/ChmB and could be purified in high yield using a simple procedure. In vitro experiments demonstrated that SgI/ChmB effectively promoted cell-substratum adhesion of NIH 3T3 and PC12 cells and supported neurite outgrowth in primary hippocampal neurons. Thus, SgI/ChmB may be a new member of the family of heparin-binding extracellular matrix proteins that mediate cell adhesion and support neurite outgrowth.     

Neurotoxicity of a Carboxyl-Terminal Fragment of the Alzheimer's Amyloid Precursor Protein

Abstract: We have previously shown that a recombinant carboxyl-terminal 105-amino-acid fragment (CT105) of the amyloid precursor protein (APP) induced strong non-selective inward currents in Xenopus oocytes. Here we investigated the toxic effect of CT105 peptide on the cultured mammalian cells. The CT105 peptide induced a significant lactate dehydrogenase (LDH) release from cultured rat cortical neurons and PC12 cells in a concentration (from 10 µ M )- and time (from 48 h)-dependent manner. The toxic effect of CT105 was more potent than that of any fragments of amyloid β protein (Aβ). However, CT105 peptide did not affect the viability of U251 human glioblastoma cells. In contrast to CT105, Aβ increased LDH release only slightly even at 50 µ M but significantly inhibited 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction at submicromolar concentrations. Among the various neuroprotective drugs tested, only cholesterol, which alters membrane fluidity, could attenuate the cytotoxicity of CT105 significantly. The CT105 peptide formed multiple self-aggregates on solubilization. Pretreatment with a sublethal concentration of CT105 did not significantly alter the susceptibility of cells to hydrogen peroxide and glutamate. Endogenous CT peptides were found not only in the cell lysates but also in the conditioned medium of PC12 cells. These results imply that CT peptide can directly attack the cell membrane probably by making pores or nonselective ion channels, whereas Aβ impairs the intracellular metabolic pathway first. Thus, it is thought that both CT and Aβ, which are formed during the processing of APP, may participate in the neuronal degeneration in Alzheimer's disease by different mechanisms.     

A 127-kDa Protein (UV-DDB) Binds to the Cytoplasmic Domain of the Alzheimer's Amyloid Precursor Protein

Abstract : Alzheimer amyloid precursor protein (APP) is an integral membrane protein with a short cytoplasmic domain of 47 amino acids. It is hoped that identification of proteins that interact with the cytoplasmic domain will provide new insights into the physiological function of APP and, in turn, into the pathogenesis of Alzheimer's disease. To identify proteins that interact with the cytoplasmic domain of APP, we employed affinity chromatography using an immobilized synthetic peptide corresponding to residues 645-694 of APP₆₉₅ and identified a protein of ~130 kDa in rat brain cytosol. Amino acid sequencing of the protein revealed the protein to be a rat homologue of monkey UV-DDB (UV-damaged DNA-binding protein, calculated molecular mass of 127 kDa). UV-DDB/p127 co-immunoprecipitated with APP using an anti-APP antibody from PC12 cell lysates. APP also co-immunoprecipitated with UV-DDB/p127 using an anti-UV-DDB/p127 antibody. These results indicate that UV-DDB/p127, which is present in the cytosolic fraction, forms a complex with APP through its cytoplasmic domain. In vitro binding experiments using a glutathione S -transferase-APP cytoplasmic domain fusion protein and several mutants indicated that the YENPTY motif within the APP cytoplasmic domain, which is important in the internalization of APP and amyloid β protein secretion, may be involved in the interaction between UV-DDB/p127 and APP.     

Amyloid Precursor Protein Mutation Disrupts Reproductive Experience-Enhanced Normal Cognitive Development in a Mouse Model of Alzheimer's Disease

Women experience dramatic changes in hormones, mood and cognition through different periods of their reproductive lives, particularly during pregnancy and giving birth. While limited human studies of early pregnancy and motherhood showed alteration of cognitive functions in later life, researches on rodents showed a persistent improvement of learning and memory performance in females with history of giving birth compared to virgin controls. Alzheimer's disease (AD), the most common dementia in elderly, is more prevalent in women than in men. One of the risk factors is related to the sharp reduction of estrogen in aged women. It is unknown whether the history of fertility activity plays any roles in altering risk of AD in females, such as altering cognitive function. Would reproductive experience alter the risk of AD in females? If so, what might be the mechanisms of the change? In this study, we examined the effects of reproductive experience on cognitive function in an AD transgenic mouse model (APP23) and age-matched wild-type non-transgenic control mice (WT). Our data showed an age-dependent effect of reproductive experience on learning and memory activity between breeders (had one or more litters) and non-breeders (virgins). More importantly, our data, for the first time, demonstrated a genotype-dependent effect of parity on cognitive function between APP23 and WT mice. At the age of 12 months, WT breeders outperform non-breeders in spatial working and reference memory while APP23 breeders performed worse in spatial learning and memory than age-matched APP23 non-breeders. These genotype- and age-dependent effects of reproductive activity on cognitions are significantly associated with changes of neuropathology of AD in the APP23 mice, expression of proteins related to synaptic plasticity and cognitive functions in the brain.     

An 82-Kilodalton Membrane Protein that Inhibits the Activity of Neurite Outgrowth Factor

Neurite outgrowth factor (NOF), an extracellular matrix glycoprotein of 700 kilodaltons (kDa), promoted neurite outgrowth from cultured ciliary ganglion (CG) neurons of chicken embryo. A fraction solubilized with Nonidet P-40 of chicken gizzard muscle membranes inhibited the neurite-promoting activity of NOF in a dose-dependent manner, but not that of laminin. Binding of CG neurons to the substratum and their survival were not affected by the extract. The inhibitory activity of the extract was abolished by treatment with trypsin or heat. The molecular size was determined to be about 82 kDa by ligand blotting. The active component was partially purified by column chromatography. It is suggested that this molecule interacts with the domain of NOF responsible for its neurite-promoting activity and may modulate NOF activity during development in vivo.     

两种构祀植物花药培养单倍体的诱导

对110例广东汉族人血清作了补体C2, Bf, C4的测定,其基因频率分V1为：C2*C'0.9500, C2*B: 0.0227,C2-,4:0182, C2*QO:O．0091;Bf*S：0．8364, Bf^`F:0．1409, Bf*S07：0．0091, Bf *S025： 0.009i,Bf*S055：0,0045; C4*A3：0.6327,C4*A4：0．1327,C4*_00：0．1020, C4*A5：0．0255 （一4*A2: 0·0918,C4*,41：0．0053;C4*B1：0．4569, C4*B2：0．4416, C4*QO:O．0558,C4*B5：0．0152,C4"}B96： 0.0152, C4*B3:0.0102, C4*B92:0.0051。木调查在我国首次发现一例C2*QO纯合子。     

Regulation of Amyloid Precursor Protein Cleavage

Abstract : Multiple lines of evidence suggest that increased production and/or deposition of the β-amyloid peptide, derived from the amyloid precursor protein, contributes to Alzheimer's disease. A growing list of neuro-transmitters, growth factors, cytokines, and hormones have been shown to regulate amyloid precursor protein processing. Although traditionally thought to be mediated by activation of protein kinase C, recent data have implicated other signaling mechanisms in the regulation of this process. Moreover, novel mechanisms of regulation involving cholesterol-, apolipoprotein E-, and stress-activated pathways have been identified. As the phenotypic changes associated with Alzheimer's disease encompass many of these signaling systems, it is relevant to determine how altered cell signaling may be contributing to increasing brain amyloid burden. We review the myriad ways in which first messengers regulate amyloid precursor protein catabolism as well as the signal transduction cascades that give rise to these effects.     

Human Brain βA4 Amyloid Protein Precursor of Alzheimer''s Disease: Purification and Partial Characterization

The major component of the amyloid deposition that characterizes Alzheimer's disease is the 4-kDa beta A4 protein, which is derived from a much larger amyloid protein precursor (APP). A procedure for the complete purification of APP from human brain is described. The same amino terminal sequence of APP was found in two patients with Alzheimer's disease and one control subject. Two major forms of APP were identified in human brain with apparent molecular masses of 100-110 kDa and 120-130 kDa. Soluble and membrane fractions of brain contained nearly equal amounts of APP in both humans and rats. Immunoprecipitation with carboxyl terminus-directed antibodies indicates that the soluble forms of APP are truncated. Carboxyl terminus truncation of membrane-associated forms of human brain APP was also found to occur during postmortem autolysis. The availability of purified human brain APP will facilitate the investigation of its normal function and the events that lead to its abnormal cleavage in patients with Alzheimer's disease.     

Modulation of Proteolytic Activity During Neuritogenesis in the PC12 Nerve Cell: Differential Control of Plasminogen Activator and Plasminogen Activator Inhibitor Activities by Nerve Growth Factor and Dibutyryl-Cyclic AMP

Extracellular proteolysis is considered to be required during neuritic outgrowth to control the adhesiveness between the growing neurite membrane and extracellular matrix proteins. In this work, PC12 nerve cells were used to study the modulation of proteolytic activity during neuronal differentiation. PC12 cells were found to contain and release a 70-75-kDa tissue-type plasminogen activator (tPA) and a much less abundant 48-kDa urokinase-type plasminogen activator. A plasminogen activator inhibitor (PAI) activity with molecular sizes of 54 and 58 kDa was also detected in PC12 cell conditioned medium and formed high-molecular-mass complexes with released tPA. Release of PAI activity was dependent on treatment with nerve growth factor (NGF), whereas tPA synthesis and release were under control of a cyclic AMP-dependent mechanism and increased on treatment with dibutyryl-cyclic AMP [(But)2cAMP] or cholera toxin. Simultaneous treatment with NGF and (But)2cAMP resulted in increases of both tPA and PAI release and enhancement of tPA-PAI complex formation. The resulting plasminogen activator activity in conditioned medium was high in (But)2cAMP-treated cultures with short neuritic outgrowth but remained low in NGF- or NGF plus (But)2cAMP-treated cultures, where neurite extension was, respectively, large and very large. These results suggest that excess proteolytic activity may be detrimental to neuritic outgrowth and that not only PAI release but also tPA-PAI complex formation is associated with production of large and stable neuritic outgrowth. This can be understood as an involvement of PAI in the protection against neurite-destabilizing proteolytic activity.     

Genetic Dissection of the Amyloid Precursor Protein in Developmental Function and Amyloid Pathogenesis

Proteolytic processing of the amyloid precursor protein (APP) generates large soluble APP derivatives, β-amyloid (Aβ) peptides, and APP intracellular domain. Expression of the extracellular sequences of APP or its Caenorhabditis elegans counterpart has been shown to be sufficient in partially rescuing the CNS phenotypes of the APP-deficient mice and the lethality of the apl-1 null C. elegans, respectively, leaving open the question as what is the role of the highly conserved APP intracellular domain? To address this question, we created an APP knock-in allele in which the mouse Aβ sequence was replaced by the human Aβ. A frameshift mutation was introduced that replaced the last 39 residues of the APP sequence. We demonstrate that the C-terminal mutation does not overtly affect APP processing and amyloid pathology. In contrast, crossing the mutant allele with APP-like protein 2 (APLP2)-null mice results in similar neuromuscular synapse defects and early postnatal lethality as compared with mice doubly deficient in APP and APLP2, demonstrating an indispensable role of the APP C-terminal domain in these development activities. Our results establish an essential function of the conserved APP intracellular domain in developmental regulation, and this activity can be genetically uncoupled from APP processing and Aβ pathogenesis.     

Structure-function analysis of Nel, a thrombospondin-1-like glycoprotein involved in neural development and functions

Nel (neural epidermal growth factor (EGF)-like molecule) is a multimeric, multimodular extracellular glycoprotein with heparin-binding activity and structural similarities to thrombospondin-1. Nel is predominantly expressed in the nervous system and has been implicated in neuronal proliferation and differentiation, retinal axon guidance, synaptic functions, and spatial learning. The Nel protein contains an N-terminal thrombospondin-1 (TSP-N) domain, five cysteine-rich domains, and six EGF-like domains. However, little is known about the functions of specific domains of the Nel protein. In this study, we have performed structure-function analysis of Nel, by using a series of expression constructs for different regions of the Nel protein. Our studies demonstrate that the TSP-N domain is responsible for homo-multimer formation of Nel and its heparin-binding activity. In vivo, Nel and related Nell1 are expressed in several regions of the mouse central nervous system with partly overlapping patterns. When they are expressed in the same cells in vitro, Nel and Nell1 can form hetero-multimers through the TSP-N domain, but they do not hetero-oligomerize with thrombospondin-1. Whereas both the TSP-N domain and cysteine-rich domains can bind to retinal axons in vivo, only the latter causes growth cone collapse in cultured retinal axons, suggesting that cysteine-rich domains interact with and activate an inhibitory axon guidance receptor. These results suggest that Nel interacts with a range of molecules through its different domains and exerts distinct functions.     

Molecular Mechanisms of Alzheimer Disease Protection by the A673T Allele of Amyloid Precursor Protein

Pathogenic mutations in the amyloid precursor protein (APP) gene have been described as causing early onset familial Alzheimer disease (AD). We recently identified a rare APP variant encoding an alanine-to-threonine substitution at residue 673 (A673T) that confers protection against development of AD (Jonsson, T., Atwal, J. K., Steinberg, S., Snaedal, J., Jonsson, P. V., Bjornsson, S., Stefansson, H., Sulem, P., Gudbjartsson, D., Maloney, J., Hoyte, K., Gustafson, A., Liu, Y., Lu, Y., Bhangale, T., Graham, R. R., Huttenlocher, J., Bjornsdottir, G., Andreassen, O. A., Jönsson, E. G., Palotie, A., Behrens, T. W., Magnusson, O. T., Kong, A., Thorsteinsdottir, U., Watts, R. J., and Stefansson, K. (2012) Nature 488, 96–99). The Ala-673 residue lies within the β-secretase recognition sequence and is part of the amyloid-β (Aβ) peptide cleavage product (position 2 of Aβ). We previously demonstrated that the A673T substitution makes APP a less favorable substrate for cleavage by BACE1. In follow-up studies, we confirm that A673T APP shows reduced cleavage by BACE1 in transfected mouse primary neurons and in isogenic human induced pluripotent stem cell-derived neurons. Using a biochemical approach, we show that the A673T substitution modulates the catalytic turnover rate (V_max) of APP by the BACE1 enzyme, without affecting the affinity (K_m) of the APP substrate for BACE1. We also show a reduced level of Aβ(1–42) aggregation with A2T Aβ peptides, an observation not conserved in Aβ(1–40) peptides. When combined in a ratio of 1:9 Aβ(1–42)/Aβ(1–40) to mimic physiologically relevant mixtures, A2T retains a trend toward slowed aggregation kinetics. Microglial uptake of the mutant Aβ(1–42) peptides correlated with their aggregation level. Cytotoxicity of the mutant Aβ peptides was not dramatically altered. Taken together, our findings demonstrate that A673T, a protective allele of APP, reproducibly reduces amyloidogenic processing of APP and also mildly decreases Aβ aggregation. These effects could together have an additive or even synergistic impact on the risk of developing AD.     

microRNA 对淀粉样前体蛋白调控的研究进展

淀粉样前体蛋白(APP)参与了神经肌肉的信号传导、突触的可塑性及空间学习等生理过程,APP在阿兹海默病(AD)人脑组织中高表达,其切割产物β淀粉样蛋白(Aβ)则在AD的发生发展中起到重要作用。2011年4月,美国阿兹海默病协会将Aβ的聚集程度列入了新版AD诊疗指南中,通过减少APP的表达或降低其以β切割方式进行代谢来延缓AD的进展已成为很多学者的共识。microRNA(miRNA)是一类内生的、长度约19-24个核苷酸的小RNA,其在细胞内具有多种重要的调节作用,据推测,miRNA调控着人类约三分之一的基因。自2008年首次明确miRNA对APP表达存在调控作用之后,miRNA对APP的调控和相关机制的研究以及其对AD诊断和治疗潜在价值的探索已成为AD研究领域的热点之一,本文主要就miRNA对APP的表达、剪切和切割的调控及Aβ对miRNA的影响做一综述。     

The Amyloid Precursor Protein (APP) Triplicated Gene Impairs Neuronal Precursor Differentiation and Neurite Development through Two Different Domains in the Ts65Dn Mouse Model for Down Syndrome

Intellectual disability in Down syndrome (DS) appears to be related to severe proliferation impairment during brain development. Recent evidence shows that it is not only cellular proliferation that is heavily compromised in DS, but also cell fate specification and dendritic maturation. The amyloid precursor protein (APP), a gene that is triplicated in DS, plays a key role in normal brain development by influencing neural precursor cell proliferation, cell fate specification, and neuronal maturation. APP influences these processes via two separate domains, the APP intracellular domain (AICD) and the soluble secreted APP. We recently found that the proliferation impairment of neuronal precursors (NPCs) from the Ts65Dn mouse model for DS was caused by derangement of the Shh pathway due to overexpression of patched1(Ptch1), its inhibitory regulator. Ptch1 overexpression was related to increased levels within the APP/AICD system. The overall goal of this study was to determine whether APP contributes to neurogenesis impairment in DS by influencing in addition to proliferation, cell fate specification, and neurite development. We found that normalization of APP expression restored the reduced neuronogenesis, the increased astrogliogenesis, and the reduced neurite length of trisomic NPCs, indicating that APP overexpression underpins all aspects of neurogenesis impairment. Moreover, we found that two different domains of APP impair neuronal differentiation and maturation in trisomic NPCs. The APP/AICD system regulates neuronogenesis and neurite length through the Shh pathway, whereas the APP/secreted AP system promotes astrogliogenesis through an IL-6-associated signaling cascade. These results provide novel insight into the mechanisms underlying brain development alterations in DS.