Iron Levels Modulate α-Secretase Cleavage of Amyloid Precursor Protein

Abstract: The amyloid precursor protein (APP) is a membrane-spanning glycoprotein that is the source of βA4 peptides, which aggregate in Alzheimer's disease to form senile plaques. APP is cleaved within the βA4 sequence to release a soluble N-terminal derivative (APP_sol), which has a wide range of trophic and protective functions. In the current study we have examined the hypothesis that iron availability may modulate expression or processing of APP, whose mRNA contains, based on sequence homology, a putative iron response element (IRE). Radiolabeled APP and its catabolites were precipitated from lysates and conditioned medium of stably transfected HEK 293 cells using antibodies selective for C-terminal, βA4, and N-terminal domains. The relative abundance of the different APP catabolites under different conditions of iron availability was determined by quantitative densitometry after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The data show a specific effect on the production of APP_sol. Using standard conditions previously established for IRE studies, it was found that iron chelation reduces APP_sol production, whereas iron level elevation augments it. No changes were observed in levels of immature and mature APP holoprotein or in the C-terminal α-secretase derivative C83, βA4, and p3 peptides. The specificity for modulatory changes in APP_sol suggests that iron acts at the level of α-secretase activity. In addition to its modulatory effects, iron at very high levels was found to inhibit maturation of APP and production of its downstream catabolites without blocking formation of immature APP. The data establish a potential physiological role for iron in controlling the processing of APP. If APP_sol were to function trophically, as suggested by other studies, the current conclusion suggests that changes in iron and iron-regulating proteins in Alzheimer's disease could contribute to neuronal degeneration by decreasing the production of APP_sol.     

An Alternative Secretase Cleavage Produces Soluble Alzheimer Amyloid Precursor Protein Containing a Potentially Amyloidogenic Sequence

Cell culture studies have shown that the Alzheimer amyloid precursor protein (APP) is secreted after full-length APP is cleaved by a putative secretase at the Lys16-Leu17 bond (secretase cleavage I) of the amyloid peptide sequence. Because this cleavage event is incompatible with amyloid production, it has been assumed that secreted APP cannot serve as a precursor of the amyloid depositions observed in Alzheimer's disease. Here we show that in neuronally differentiated PC12 cells and human kidney 293 cell cultures a portion of the secreted extracytoplasmic APP reacted specifically with both a monoclonal antibody recognizing amyloid protein residues Leu17-Val24 and a polyclonal antiserum directed against amyloid protein residues Ala21-Lys28. Furthermore, this APP failed to react with antisera recognizing the cytoplasmic domain of the full-length protein. These data indicate the presence of an alternative APP secretase cleavage site (secretase cleavage II), C-terminal to the predominant secretase cleavage I. Depending on the exact location of cleavage site II, potentially amyloidogenic secreted APP species may be produced.     

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.     

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.     

Evidence Against a Role for the Kunitz Domain in Amyloidogenic and Secretory Processing of the Amyloid Precursor Protein

Abstract: The effect of the Kunitz proteinase inhibitor (KPI) on potential β-amyloid precursor protein (βPP)-processing activities from control and Alzheimer's disease (AD) brains was examined using fluorogenic substrates designed to mimic the secretory and amyloidogenic cleavages in βPP. In addition, the level of secretion of KPI-containing βPP751 and KPI-lacking βPP695 from transfected cells was examined to assess the effect of the KPI on βPP secretion. βPP751 and βPP695, obtained from conditioned media of transfected cells, had no effect on proteinase activities against the secretory and amyloidogenic substrates in extracts from control and AD brains. At similar concentrations βPP751, but not βPP695, completely inhibited the activity of trypsin against these substrates. Serine proteinase inhibitors had only modest effects on activities from brain, whereas cysteine modification completely inhibited them, indicating that these proteinase activities were not of the serine type. Thus, the results do not support a role for the KPI in the secretion of βPP or in the amyloidogenic cleavage of βPP. The amounts of βPP695 and βPP751 collected from the media of transfected cells after 48 h of growth were similar, indicating an equal rate of secretion. This result suggests that the KPI domain in βPP751 did not inhibit the secretory cleavage in transfected cells.     

Galanin Inhibits Noradrenaline-Induced Accumulation of Cyclic AMP in the Rat Cerebral Cortex

The effect of galanin on noradrenaline (NA)-induced accumulation of cyclic AMP was investigated in slices of rat cerebral cortex. NA (10(-4)M) increased cyclic AMP levels during a 20-min observation period. Galanin (3 X 10(-7)M) significantly inhibited this response at all time points examined, although it did not change the basal levels of cyclic AMP. Galanin (10(-8)-3 X 10(-6)M) inhibited the cyclic AMP response to NA (10(-4)M) in a dose-dependent manner, with an IC50 of approximately 5.6 X 10(-8)M and a maximum inhibition of 59%. These results suggest that galanin, devoid of any detectable effects by itself, modulates the cyclic AMP response to NA in the rat cerebral cortex.     

Intracellular pH on Protein Kinase C and Ionomycin Potentiation of Isoproterenol-Stimulated Cyclic AMP and Cyclic GMP Production in Rat Pinealocytes

In rat pinealocytes, alpha 1-adrenergic activation, which leads to cytoplasmic alkalinization, also potentiates the beta-adrenergic stimulated cyclic AMP (cAMP) and cyclic GMP (cGMP) responses. Both elevation of intracellular calcium ([Ca2+]i) and activation of protein kinase C are involved in the potentiation mechanism. Recently, intracellular pH has also been found to modulate the adrenergic-stimulated cyclic nucleotide responses, suggesting intracellular pH may also affect the potentiation mechanism. This possibility was examined in the present study. Cytoplasmic alkalinization by ammonium chloride had an enhancing effect on the isoproterenol and ionomycin-stimulated cAMP and cGMP accumulation. In comparison, cytoplasmic acidification by sodium propionate reduced the isoproterenol and ionomycin-stimulated cAMP and cGMP responses. Direct measurement of [Ca2+]i indicated that neither ammonium chloride nor sodium propionate had an effect on the ionomycin-stimulated elevation of [Ca2+]i, suggesting their effects on cyclic nucleotide responses may be independent of [Ca2+]i. In cells stimulated by isoproterenol and an activator of protein kinase C, ammonium chloride had an enhancing effect on both cAMP and cGMP responses, whereas sodium propionate had no effect. Taken together, these results suggest that a site distal to elevation of [Ca2+]i and activation of protein kinase C, of importance to the potentiation mechanism, is modulated by intracellular pH.     

Endosomal Sorting of Amyloid Precursor Protein-P-Selectin Chimeras Influences Secretase Processing

Amyloid β protein, the major component of the senile plaques in Alzheimer's disease, is generated by secretory and endocytic processing of amyloid precursor protein. Internalized amyloid precursor protein either recycles to the plasma membrane, where α-secretase resides, or moves to acidic compartment(s) for β-secretase exposure. While the trans-Golgi network contains β-secretase activity, recent examination of the subcellular distribution of this proteinase, called BACE, has led to the suggestion that β-secretase activity might also reside at the plasma membrane and in endosomes. To examine the role of endocytic compartments in β-secretase processing of amyloid precursor protein, the wild-type and endosomal sorting mutant P-selectin cytoplasmic domains were used to control movement of amyloid precursor protein through endosomes. Amyloid precursor protein/P-selectin, which is sorted from early to late endosomes, undergoes significantly less α-secretase cleavage, and more β-secretase cleavage, than amyloid precursor protein/P-selectin768A, a mutant that recycles more efficiently to the cell surface. Our results demonstrate that endosomal sorting influences relative exposure of the amyloid precursor protein/P-selectin chimeras to α- and β-secretase activities, and suggest that, because delivery to late endocytic compartments favors β-secretase processing of amyloid precursor protein, there is likely limited β-secretase activity in early endosomes or at the cell surface. We propose that the trans-Golgi network may be involved in both secretory and endocytic generation of amyloid β protein.     

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。     

Muscarinic Receptor Stimulation Increases Inositol-Phospholipid Metabolism and Inhibits Cyclic AMP Accumulation in PC 12 Cells

Muscarinic receptor stimulation increased the accumulation of 3H-inositol phosphates in PC12 cells whose phospholipids had been prelabeled with [3H]inositol. Muscarine also inhibited the increase in cyclic AMP (cAMP) accumulation caused by 5'-N-ethylcarboxamide adenosine or by vasoactive intestinal peptide. This effect of muscarine was apparently due to the inhibition of adenylate cyclase rather than to a stimulation of a cAMP specific phosphodiesterase. The muscarinic receptor antagonist pirenzepine inhibited both the stimulation of inositol-phospholipid metabolism and the inhibition of cAMP production with Ki values of 0.34 microM and 0.36 microM, respectively. PC12 cells contained a single class of N-[3H]methylscopolamine ([3H]NMS) binding sites. Competition studies with muscarine (KD, 15 microM) and pirenzepine (Ki, 0.12 microM) revealed no evidence for multiple muscarinic receptors. The Ki of pirenzepine for the inhibition of [3H]NMS binding and the inhibition of muscarinic actions is consistent with the possibility that this is not an M1 receptor. Muscarine inhibited cAMP accumulation in cells made deficient in protein kinase C; therefore, this protein kinase is probably not involved in mediating the inhibitory effect of muscarine. The phorbol ester 12-O-tetradecanoylphorbol 13-acetate also inhibited cAMP accumulation in PC12 cells but the mechanism of this effect differed from that of muscarine. Bradykinin caused a large increase in the accumulation of 3H-inositol phosphates and [3H]diacylglycerol relative to muscarine but did not inhibit cAMP production. Oxotremorine inhibited cAMP accumulation but it did not stimulate inositol-phospholipid metabolism.(ABSTRACT TRUNCATED AT 250 WORDS)     

Increased Secretion of the Amino-Terminal Fragment of Amyloid Precursor Protein in Brains of Rats with a Constitutive Up-Regulation of Protein Kinase C

Abstract: Protein kinase C (PKC) activation stimulates release of secreted amyloid precursor protein (APP_s) in several cell lines. To ascertain the role of PKC in regulating APP metabolism in vivo, we used an animal model (methylazoxymethanol-treated rats; MAM rats) in which PKC is permanently hyperactivated in selected brain areas, i.e., cortex and hippocampus. A significant decrease in membrane-bound APP concentration was found in synaptosomes derived from cortex and hippocampus of MAM rats, where PKC is up-regulated, with a concomitant increase in APP_s production in soluble fractions of the same brain areas. In contrast, in a brain area not affected by MAM treatment (i.e., cerebellum), APP secretion is similar in control and MAM rats, indicating that altered metabolism of APP is restricted to only those areas in which the PKC system is up-regulated. In addition, phorbol esters or H-7 modulate APP_s release in hippocampal slices from both control and MAM rats, further supporting an in vivo role for this enzyme in regulating metabolism of mature APP.     

Novel Adaptors of Amyloid Precursor Protein Intracellular Domain and Their Functional Implications

Amyloid precursor protein intracellular domain(AICD) is one of the potential candidates in deciphering the complexity of Alzheimer’s disease.It plays important roles in determining cell fate and neurodegeneration through its interactions with several adaptors.The presence or absence of phosphorylation at specific sites determines the choice of partners.In this study,we identified 20 novel AICDinteracting proteins by in vitro pull down experiments followed by 2D gel electrophoresis and MALDI-MS analysis.The identified proteins can be grouped into different functional classes including molecular chaperones,structural proteins,signaling and transport molecules,adaptors,motor proteins and apoptosis determinants.Interactions of nine proteins were further validated either by colocalization using confocal imaging or by co-immunoprecipitation followed by immunoblotting.The cellular functions of most of the proteins can be correlated with AD.Hence,illustration of their interactions with AICD may shed some light on the disease pathophysiology.     

Cyclic AMP Inhibits Activation of Mitogen-Activated Protein Kinase and Cell Proliferation in Response to Growth Factors in Cultured Rat Cortical Astrocytes

Abstract: The cyclic AMP (cAMP)-induced inhibitory effect on cell proliferation was examined through inhibition of mitogen-activated protein kinase (MAP kinase) activation in cultured rat cortical astrocytes. Basic fibroblast growth factor (bFGF) at 10 ng/ml maximally stimulated MAP kinase activity, which peaks during 10 min and prolonged for 24 h. Likewise, DNA synthesis was maximally potentiated with 10 ng/ml bFGF and correlated with MAP kinase activity in a dose-dependent manner. Dibutyryl cAMP (dbcAMP) at 1 m M and isoproterenol at 10 µ M inhibited MAP kinase activation and DNA synthesis potentiation with bFGF and platelet-derived growth factor to the control level in cultured astrocytes and C6 glioma cells. The stimulation with bFGF caused a prominent translocation of MAP kinase from the cytosol to the nucleus after 1 h in astrocytes. Treatment of the cells with dbcAMP and isoproterenol completely prevented the translocation of MAP kinase. In experiments with ³²P-labeled cultured astrocytes, phosphorylation of Raf-1 was apparently stimulated with bFGF. Treatment with dbcAMP or isoproterenol had a greatly inhibitory effect on the stimulation of Raf-1 phosphorylation with bFGF. Consistent with the effect on Raf-1 phosphorylation, dbcAMP and isoproterenol completely prevented bFGF-induced phosphorylation of MAP kinase kinases, target proteins of Raf-1. Our observations suggest that cAMP-induced suppression of cell growth in astrocytes is due to the inhibitory effect on activation of MAP kinase and its translocation to the nucleus and that the site of the cAMP action is located at Raf-1 or the upstream site of Raf-1.     

Modulation of Intracellular Cyclic AMP Levels by Different Human Dopamine D4 Receptor Variants

Abstract: To investigate whether polymorphic forms of the human dopamine D4 receptor have different functional characteristics, we have stably expressed cDNAs of the D4.2, D4.4, and D4.7 isoforms in several cell lines. Chinese hamster ovary CHO-K1 cell lines expressing D4 receptor variants displayed pharmacological profiles that were in close agreement with previous data from transiently expressed D4 receptors in COS-7 cells. Dopamine stimulation of the D4 receptors resulted in a concentration-dependent inhibition of the forskolin-stimulated cyclic AMP (cAMP) levels. The potency of dopamine to inhibit cAMP formation was about twofold reduced for D4.7 (EC₅₀ of ∼37 n M ) compared with the D4.2 and D4.4 variants (EC₅₀ of ∼16 n M ). Antagonists block the dopamine-mediated inhibition of cAMP formation with a rank order of potency of emonapride > haloperidol = clozapine ≫ raclopride. There was no obvious correlation between the efficacy of inhibition of forskolin-stimulated cAMP levels and the D4 subtypes. Dopamine could completely reverse prostaglandin E₂-stimulated cAMP levels for all three D4 receptor variants. Deletion of the repeat sequence does not affect functional activity of the receptor. The data presented indicate that the polymorphic repeat sequence causes only small changes in the ability of the D4 receptor to block cAMP production in CHO cells.     

Phorbol Esters Enhance Neurotransmitter-Stimulated Cyclic AMP Production in Rat Brain Slices

The effect of phorbol esters on cyclic AMP production in rat CNS tissue was examined. Using a prelabeling technique for measuring cyclic AMP accumulation in brain slices, it was found that phorbol 12-myristate, 13-acetate (PMA) enhanced the cyclic AMP response to forskolin and a variety of neurotransmitter receptor stimulants while having no effect on second messenger accumulation itself. A short (15-min) preincubation period with PMA was required to obtain maximal enhancement, whereas the augmentation was lessened by prolonged exposure (3 h) to the phorbol. The response to PMA was concentration dependent (EC50 = 1 microM) and regionally selective, being most apparent in forebrain, and was not influenced by removal of extracellular calcium or by inhibition of phosphodiesterase or phospholipase A2. Only those phorbols known to stimulate protein kinase C augmented the accumulation of cyclic AMP. Moreover, the membrane substrates phosphorylated by endogenous C kinase and by a partially purified preparation of this enzyme were similar. The results suggest that phorbol esters, by activating protein kinase C, modify the cyclic AMP response to brain neurotransmitter receptor stimulation in brain by influencing a component of the adenylate cyclase system beyond the transmitter recognition site.     

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.