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1.
The amyloid precursor protein (APP) is proteolytically processed predominantly by alpha-secretase to release the ectodomain (sAPPalpha). In this study, we have addressed the cellular location of the constitutive alpha-secretase cleavage of endogenous APP in a neuronal cell line. Incubation of the neuroblastoma cell line IMR32 at 20 degrees C prevented the secretion into the medium of soluble wild-type APP cleaved by alpha-secretase as revealed by both immunoelectrophoretic blot analysis with a site-specific antibody and immunoprecipitation following metabolic labeling of the cells. No sAPPalpha was detected in the cell lysates following incubation of the cells at 20 degrees C, indicating that alpha-secretase does not cleave APP in the secretory pathway prior to or within the trans-Golgi network. Parallel studies using an antibody that recognizes specifically the neoepitope revealed on soluble APP cleaved by beta-secretase indicated that this enzyme was acting intracellularly. alpha-Secretase is a zinc metalloproteinase susceptible to inhibition by hydroxamate-based compounds such as batimastat [Parvathy, S., et al. (1998) Biochemistry 37, 1680-1685]. Incubation of the cells with a cell-impermeant, biotinylated hydroxamate inhibitor inhibited the release of sAPPalpha by >92%, indicating that alpha-secretase is cleaving APP almost exclusively at the cell surface. The observation that alpha-secretase cleaves APP at the cell surface, while beta-secretase can act earlier in the secretory pathway within the neuronal cell line indicates that there must be strict control mechanisms in place to ensure that APP is normally cleaved primarily by alpha-secretase in the nonamyloidogenic pathway to produce the neuroprotective sAPPalpha.  相似文献   

2.
3.
The metabolism of the amyloid precursor protein (APP) has been extensively investigated because its processing generates the amyloid-β-peptide (Aβ), which is a likely cause of Alzheimer disease. Much prior research has focused on APP processing using transgenic constructs and heterologous cell lines. Work to date in native neuronal cultures suggests that Aβ is produced in very large amounts. We sought to investigate APP metabolism and Aβ production simultaneously under more physiological conditions in vivo and in vitro using cultured rat cortical neurons and live pigs. We found in cultured neurons that both APP and Aβ are secreted rapidly and at extremely high rates into the extracellular space (2-4 molecules/neuron/s for Aβ). Little APP is degraded outside of the pathway that leads to extracellular release. Two metabolic pools of APP are identified, one that is metabolized extremely rapidly (t1/2;) = 2.2 h), and another, surface pool, composed of both synaptic and extrasynaptic elements, that turns over very slowly. Aβ release and accumulation in the extracellular medium can be accounted for stoichiometrically by the extracellular release of β-cleaved forms of the APP ectodomain. Two α-cleavages of APP occur for every β-cleavage. Consistent with the results seen in cultured neurons, an extremely high rate of Aβ production and secretion from the brain was seen in juvenile pigs. In summary, our experiments show an enormous and rapid production and extracellular release of Aβ and the soluble APP ectodomain. A small, slowly metabolized, surface pool of full-length APP is also identified.  相似文献   

4.
The beta-amyloid precursor protein (APP)-binding protein Fe65 is involved in APP nuclear signaling and several steps in APP proteolytic processing. In this study, we show that Fe65 stimulates gamma-secretase-mediated liberation of the APP intracellular domain (AICD). The mechanism of Fe65-mediated stimulation of AICD formation appears to be through enhanced production of the carboxyl-terminal fragment substrates of gamma-secretase and direct stimulation of processing by gamma-secretase. The stimulatory capacity of Fe65 is isoform-dependent, as the non-neuronal and a2 isoforms promote APP processing more effectively than the exon 9 inclusive neuronal form of Fe65. Intriguingly, Fe65 stimulation of AICD production appears to be inversely related to pathogenic beta-amyloid production as the Fe65 isoforms profoundly stimulate AICD production and simultaneously decrease Abeta42 production. Despite the capacity of Fe65 to stimulate gamma-secretase-mediated APP proteolysis, it does not rescue the loss of proteolytic function associated with the presenilin-1 familial Alzheimer disease mutations. These data suggest that Fe65 regulation of APP proteolysis may be integrally associated with its nuclear signaling function, as all antecedent proteolytic steps prior to release of Fe65 from the membrane are fostered by the APP-Fe65 interaction.  相似文献   

5.
The alternative routes of cleavage of the amyloid precursor protein (APP) result in the generation and secretion of both soluble APP and beta-amyloid, the latter being the main component of the amyloid deposits in the brains of individuals with Alzheimer's disease (AD). This study examined the question of whether acetylcholinesterase (AChE) inhibitors can alter the processing of APP and the level of protein kinase C (PKC) in primary rat basal forebrain cultures. Western blotting was used to test two AChE inhibitors (reversible and irreversible) for their ability to enhance the release of APP and PKC content. These inhibitors were ambenonium (AMB) and metrifonate (MTF), at different concentrations. A significant increase was found in the cell-associated APP level in a basal forebrain neuronal culture, and there was an elevation of the APP release into the medium. Increases were similarly observed in the PKC levels after AMB or MTF treatment. The results suggest that these AChE inhibitors promote the non-amyloidogenic route of APP processing, which may be due to their stimulatory effects on PKC. The PKC activation may enhance the alpha-secretase activity and consequently the production of the N-terminal APP. Since both a decreased level of APP secretion and a low activity and level of PKC may be involved in the pathogenesis of AD, it is concluded that the administration of AChE inhibitors to AD patients may facilitate the memory processes and exert a neuroprotective effect.  相似文献   

6.
Most Alzheimer disease (AD) patients show deposition of amyloid β (Aβ) peptide in blood vessels as well as the brain parenchyma. We previously found that vascular endothelial cells express amyloid β precursor protein (APP) 770, a different APP isoform from neuronal APP695, and produce Aβ. Since the soluble APP cleavage product, sAPP, is considered to be a possible marker for AD diagnosis, sAPP has been widely measured as a mixture of these variants. We hypothesized that measurement of the endothelial APP770 cleavage product in patients separately from that of neuronal APP695 would enable discrimination between endothelial and neurological dysfunctions. Using our newly developed ELISA system for sAPP770, we observed that inflammatory cytokines significantly enhanced sAPP770 secretion by endothelial cells. Furthermore, we unexpectedly found that sAPP770 was rapidly released from activated platelets. We also found that cerebrospinal fluid mainly contained sAPP695, while serum mostly contained sAPP770. Finally, to test our hypothesis that sAPP770 could be an indicator for endothelial dysfunction, we applied our APP770 ELISA to patients with acute coronary syndrome (ACS), in which endothelial injury and platelet activation lead to fibrous plaque disruption and thrombus formation. Development of a biomarker is essential to facilitate ACS diagnosis in clinical practice. The results revealed that ACS patients had significantly higher plasma sAPP770 levels. Furthermore, in myocardial infarction model rats, an increase in plasma sAPP preceded the release of cardiac enzymes, currently used markers for acute myocardial infarction. These findings raise the possibility that sAPP770 can be a useful biomarker for ACS.  相似文献   

7.
Abstract: S100β has been implicated in the formation of dystrophic neurites, overexpressing β-amyloid precursor protein (βAPP), in the β-amyloid plaques of Alzheimer's disease. We assessed the effects of S100β on cell viability of, neurite outgrowth from, and βAPP expression by neurons in primary cultures from fetal rat cortex. S100β (1–10 ng/ml) enhanced neuronal viability (as assessed by increased mitochondrial activity and decreased lactic acid dehydrogenase release) and promoted neurite outgrowth. Higher levels of S100β (100 ng/ml, but not 1 µg/ml) produced qualitatively similar, but less marked, effects. S100β also induced increased neuronal expression of the microtubule-associated protein MAP2, an effect that is consistent with trophic effects of S100β on neurite outgrowth. S100β (10 and 100 ng/ml) induced graded increases in neuronal expression of βAPP and of βAPP mRNA. These results support our previous suggestion that excessive expression of S100β by activated, plaque-associated astrocytes in Alzheimer's disease contributes to the appearance of dystrophic neurites overexpressing βAPP in diffuse amyloid deposits, and thus to the conversion of these deposits into the diagnostic neuritic β-amyloid plaques.  相似文献   

8.
Mutant presenilin-1 (PS1) increases amyloid peptide production, attenuates capacitative calcium entry (CCE), and augments calcium release from the endoplasmatic reticulum (ER). Here we measured the intracellular free Ca(2+) concentration in hippocampal neurons from six different combinations of transgenic and gene-ablated mice to demonstrate that mutant PS1 attenuated CCE directly, independent of the expression of the amyloid precursor protein (APP). On the other hand, increased Ca(2+) release from the ER in mutant PS1 neurons, as induced by thapsigargin, was clearly dependent on the presence of APP and its processing by PS1, i.e. on the generation of the amyloid peptides and the APP C99 fragments. This observation was corroborated by the thapsigargin-induced increase in cytosolic [Ca(2+)](i) in PS1 deficient neurons, which accumulate C99 fragments due to deficient gamma-secretase activity. Moreover, co-expression of mutant APP[V717I] in PS1-deficient neurons further increased the apparent size of the ER calcium stores in parallel with increasing levels of the APP processing products. We conclude that mutant PS1 deregulates neuronal calcium homeostasis by two different actions: (i) direct attenuation of CCE at the cell-surface independent of APP; and (ii) indirect increase of ER-calcium stores via processing of APP and generation of amyloid peptides and C99 fragments.  相似文献   

9.
Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the presence in the brain of senile plaques which contain an amyloid core made of beta-amyloid peptide (Abeta). Abeta is produced by the cleavage of the amyloid precursor protein (APP). Since impairment of neuronal calcium signalling has been causally implicated in ageing and AD, we have investigated the influence of an influx of extracellular calcium on the metabolism of human APP in rat cortical neurones. We report that a high cytosolic calcium concentration, induced by neuronal depolarization, inhibits the alpha-secretase cleavage of APP and triggers the accumulation of intraneuronal C-terminal fragments produced by the beta-cleavage of the protein (CTFbeta). Increase in cytosolic calcium concentration specifically induces the production of large amounts of intraneuronal Abeta1-42, which is inhibited by nimodipine, a specific antagonist of l-type calcium channels. Moreover, calcium release from endoplasmic reticulum is not sufficient to induce the production of intraneuronal Abeta, which requires influx of extracellular calcium mediated by the capacitative calcium entry mechanism. Therefore, a sustained high concentration of cytosolic calcium is needed to induce the production of intraneuronal Abeta1-42 from human APP. Our results show that this accumulation of intraneuronal Abeta1-42 induces neuronal death, which is prevented by a functional gamma-secretase inhibitor.  相似文献   

10.
Comorbid depression of Alzheimer's disease (AD) is a common mood disorder in the elderly and a broad spectrum of antidepressants have been used for its treatment. Abeta peptides and other derivatives of the amyloid precursor protein (APP) have been implicated as central to the pathogenesis of AD. However, the functional relationship of APP and its proteolytic derivatives to antidepressant therapy is not known. In this study, Western blotting was used to test the ability of the tricyclic antidepressant (TCA) imipramine or the selective serotonin reuptake inhibitor (SSRI) citalopram to change the release of APP and the protein kinase C (PKC) content. Both antidepressants increased APP secretion in primary rat neuronal cultures. Imipramine or citalopram enhanced the level of secreted APP by 3.2- or 3.4-fold, respectively. Increases in PKC level were observed only after imipramine treatment. These in vitro data suggest that both TCA and SSRI are able to interfere with the APP metabolism. Imipramine promotes the non-amyloidogenic route of APP processing via stimulatory effects on PKC. We propose that PKC is not involved in the mechanism underlying the effects of citalopram on the APP metabolism. Since the secreted APP is not further available for the pathological cleavage of beta- and gamma-secretases, antidepressant medication might be beneficial in AD therapy.  相似文献   

11.
We reported that iron influx drives the translational expression of the neuronal amyloid precursor protein (APP), which has a role in iron efflux. This is via a classic release of repressor interaction of APP mRNA with iron-regulatory protein-1 (IRP1) whereas IRP2 controls the mRNAs encoding the L- and H-subunits of the iron storage protein, ferritin. Here, we identified thirteen potent APP translation blockers that acted selectively towards the uniquely configured iron-responsive element (IRE) RNA stem loop in the 5′ untranslated region (UTR) of APP mRNA. These agents were 10-fold less inhibitory of 5′UTR sequences of the related prion protein (PrP) mRNA. Western blotting confirmed that the ‘ninth’ small molecule in the series selectively reduced neural APP production in SH-SY5Y cells at picomolar concentrations without affecting viability or the expression of α-synuclein and ferritin. APP blocker-9 (JTR-009), a benzimidazole, reduced the production of toxic Aβ in SH-SY5Y neuronal cells to a greater extent than other well tolerated APP 5′UTR-directed translation blockers, including posiphen, that were shown to limit amyloid burden in mouse models of Alzheimer''s disease (AD). RNA binding assays demonstrated that JTR-009 operated by preventing IRP1 from binding to the IRE in APP mRNA, while maintaining IRP1 interaction with the H-ferritin IRE RNA stem loop. Thus, JTR-009 constitutively repressed translation driven by APP 5′UTR sequences. Calcein staining showed that JTR-009 did not indirectly change iron uptake in neuronal cells suggesting a direct interaction with the APP 5′UTR. These studies provide key data to develop small molecules that selectively reduce neural APP and Aβ production at 10-fold lower concentrations than related previously characterized translation blockers. Our data evidenced a novel therapeutic strategy of potential impact for people with trisomy of the APP gene on chromosome 21, which is a phenotype long associated with Down syndrome (DS) that can also cause familial Alzheimer''s disease.  相似文献   

12.
Expression of the genes encoding the beta/A4 amyloid protein precursor (APP) and microtubule-associated protein tau was studied in an embryonal carcinoma cell line (P19) that differentiates in vitro into cholinergic neurons after treatment with retinoic acid. Expression of APP increased 34- (mRNA) and 50-fold (protein) during neuronal differentiation; APP-695 accounted for most of this increase. These remarkable increases in APP expression coincided with a proliferation of neuronal processes and with an increase in content of tau mRNA. Moreover, subsequent decreases in the levels of APP and tau mRNA coincided with the onset of the degeneration of the neuronal processes. Immunocytochemical staining suggested that greater than 85% of the P19-derived neurons are cholinergic and that APP is present in the neuronal processes and cell bodies. These results suggest that APP may play an important role in construction of neuronal networks and neuronal differentiation and also indicate that this embryonal carcinoma cell line provides an ideal model system to investigate biological functions of APP and the roles of APP and tau protein in development of Alzheimer's disease in cholinergic neurons.  相似文献   

13.
APP processing and synaptic function   总被引:39,自引:0,他引:39  
A large body of evidence has implicated Abeta peptides and other derivatives of the amyloid precursor protein (APP) as central to the pathogenesis of Alzheimer's disease (AD). However, the functional relationship of APP and its proteolytic derivatives to neuronal electrophysiology is not known. Here, we show that neuronal activity modulates the formation and secretion of Abeta peptides in hippocampal slice neurons that overexpress APP. In turn, Abeta selectively depresses excitatory synaptic transmission onto neurons that overexpress APP, as well as nearby neurons that do not. This depression depends on NMDA-R activity and can be reversed by blockade of neuronal activity. Synaptic depression from excessive Abeta could contribute to cognitive decline during early AD. In addition, we propose that activity-dependent modulation of endogenous Abeta production may normally participate in a negative feedback that could keep neuronal hyperactivity in check. Disruption of this feedback system could contribute to disease progression in AD.  相似文献   

14.
Alzheimer's disease is a progressive neurodegenerative disorder characterized by extracellular deposition of beta-amyloid (Abeta) peptide containing neuritic plaques. Abeta peptides are proteolytically derived from the membrane-bound amyloid precursor protein (APP). Although the function of APP is not entirely clear, previous studies demonstrate that neuronal APP colocalizes with beta(1) integrin receptors at sites of focal adhesion, suggesting that APP is involved in mediating neuronal process adhesion. Integrin-dependent adhesion is also a well-characterized component of immune cell proinflammatory activation. Using primary mouse microglia and the human monocytic cell line, THP-1, we have begun investigating the role of APP in integrin-dependent activation. Co-immunoprecipitation studies demonstrate that APP is recruited into a multi-receptor signaling complex during beta(1) integrin-mediated adhesion of monocytes. Stimulation induces a subsequent, specific recruitment of tyrosine phosphorylated proteins to APP, including Lyn and Syk. Antibody cross-linking of cell surface APP leads to a similar response characterized by activation and recruitment of tyrosine kinases to APP as well as subsequent activation of mitogen-activated protein kinases and increased proinflammatory protein levels. These data demonstrate that APP can act as a proinflammatory receptor in monocytic lineage cells and provide insight into the contribution of this protein to the inflammatory conditions described in Alzheimer's disease.  相似文献   

15.
A major source of free radical production in the brain derives from copper. To prevent metal-mediated oxidative stress, cells have evolved complex metal transport systems. The Alzheimer's disease amyloid precursor protein (APP) is a major regulator of neuronal copper homeostasis. APP knockout mice have elevated copper levels in the cerebral cortex, whereas APP-overexpressing transgenic mice have reduced brain copper levels. Importantly, copper binding to APP can greatly reduce amyloid beta production in vitro. To understand this interaction at the molecular level we solved the structure of the APP copper binding domain (CuBD) and found that it contains a novel copper binding site that favors Cu(I) coordination. The surface location of this site, structural homology of CuBD to copper chaperones, and the role of APP in neuronal copper homeostasis are consistent with the CuBD acting as a neuronal metallotransporter.  相似文献   

16.
Alzheimer disease (AD), the most frequent cause of dementia, is characterized by an important neuronal loss. A typical histological hallmark of AD is the extracellular deposition of beta-amyloid peptide (A beta), which is produced by the cleavage of the amyloid precursor protein (APP). Most of the gene mutations that segregate with the inherited forms of AD result in increasing the ratio of A beta 42/A beta 40 production. A beta 42 also accumulates in neurons of AD patients. Altogether, these data strongly suggest that the neuronal production of A beta 42 is a critical event in AD, but the intraneuronal A beta 42 toxicity has never been demonstrated. Here, we report that the long term expression of human APP in rat cortical neurons induces apoptosis. Although APP processing leads to production of extracellular A beta 1-40 and soluble APP, these extracellular derivatives do not induce neuronal death. On the contrary, neurons undergo apoptosis as soon as they accumulate intracellular A beta 1-42 following the expression of full-length APP or a C-terminal deleted APP isoform. The inhibition of intraneuronal A beta 1-42 production by a functional gamma-secretase inhibitor increases neuronal survival. Therefore, the accumulation of intraneuronal A beta 1-42 is the key event in the neurodegenerative process that we observed.  相似文献   

17.
Amyloid precursor protein (APP), the precursor of Abeta, has been shown to function as a cell surface receptor that mediates neuronal cell death by anti-APP antibody. The c-Jun N-terminal kinase (JNK) can mediate various neurotoxic signals, including Abeta neurotoxicity. However, the relationship of APP-mediated neurotoxicity to JNK is not clear, partly because APP cytotoxicity is Abeta independent. Here we examined whether JNK is involved in APP-mediated neuronal cell death and found that: (i) neuronal cell death by antibody-bound APP was inhibited by dominant-negative JNK, JIP-1b and SP600125, the specific inhibitor of JNK, but not by SB203580 or PD98059; (ii) constitutively active (ca) JNK caused neuronal cell death and (iii) the pharmacological profile of caJNK-mediated cell death closely coincided with that of APP-mediated cell death. Pertussis toxin (PTX) suppressed APP-mediated cell death but not caJNK-induced cell death, which was suppressed by Humanin, a newly identified neuroprotective factor which inhibits APP-mediated cytotoxicity. In the presence of PTX, the PTX-resistant mutant of Galphao, but not that of Galphai, recovered the cytotoxic action of APP. These findings demonstrate that JNK is involved in APP-mediated neuronal cell death as a downstream signal transducer of Go.  相似文献   

18.
Aberrant amyloid β (Aβ) production plays a causal role in Alzheimer disease pathogenesis. A major cellular pathway for Aβ generation is the activity-dependent endocytosis and proteolytic cleavage of the amyloid precursor protein (APP). However, the molecules controlling activity-dependent APP trafficking in neurons are less defined. Mints are adaptor proteins that directly interact with the endocytic sorting motif of APP and are functionally important in regulating APP endocytosis and Aβ production. We analyzed neuronal cultures from control and Mint knockout neurons that were treated with either glutamate or tetrodotoxin to stimulate an increase or decrease in neuronal activity, respectively. We found that neuronal activation by glutamate increased APP endocytosis, followed by elevated APP insertion into the cell surface, stabilizing APP at the plasma membrane. Conversely, suppression of neuronal activity by tetrodotoxin decreased APP endocytosis and insertion. Interestingly, we found that activity-dependent APP trafficking and Aβ generation were blocked in Mint knockout neurons. We showed that wild-type Mint1 can rescue APP internalization and insertion in Mint knockout neurons. In addition, we found that Mint overexpression increased excitatory synaptic activity and that APP was internalized predominantly to endosomes associated with APP processing. We demonstrated that presenilin 1 (PS1) endocytosis requires interaction with the PDZ domains of Mint1 and that this interaction facilitates activity-dependent colocalization of APP and PS1. These findings demonstrate that Mints are necessary for activity-induced APP and PS1 trafficking and provide insight into the cellular fate of APP in endocytic pathways essential for Aβ production.  相似文献   

19.
Bailey JA  Ray B  Greig NH  Lahiri DK 《PloS one》2011,6(7):e21954
Overproduction of amyloid-β (Aβ) protein in the brain has been hypothesized as the primary toxic insult that, via numerous mechanisms, produces cognitive deficits in Alzheimer's disease (AD). Cholinesterase inhibition is a primary strategy for treatment of AD, and specific compounds of this class have previously been demonstrated to influence Aβ precursor protein (APP) processing and Aβ production. However, little information is available on the effects of rivastigmine, a dual acetylcholinesterase and butyrylcholinesterase inhibitor, on APP processing. As this drug is currently used to treat AD, characterization of its various activities is important to optimize its clinical utility. We have previously shown that rivastigmine can preserve or enhance neuronal and synaptic terminal markers in degenerating primary embryonic cerebrocortical cultures. Given previous reports on the effects of APP and Aβ on synapses, regulation of APP processing represents a plausible mechanism for the synaptic effects of rivastigmine. To test this hypothesis, we treated degenerating primary cultures with rivastigmine and measured secreted APP (sAPP) and Aβ. Rivastigmine treatment increased metabolic activity in these cultured cells, and elevated APP secretion. Analysis of the two major forms of APP secreted by these cultures, attributed to neurons or glia based on molecular weight showed that rivastigmine treatment significantly increased neuronal relative to glial secreted APP. Furthermore, rivastigmine treatment increased α-secretase cleaved sAPPα and decreased Aβ secretion, suggesting a therapeutic mechanism wherein rivastigmine alters the relative activities of the secretase pathways. Assessment of sAPP levels in rodent CSF following once daily rivastigmine administration for 21 days confirmed that elevated levels of APP in cell culture translated in vivo. Taken together, rivastigmine treatment enhances neuronal sAPP and shifts APP processing toward the α-secretase pathway in degenerating neuronal cultures, which mirrors the trend of synaptic proteins, and metabolic activity.  相似文献   

20.
The female sex hormone estrogen (17beta-estradiol; E2) may function as a neurohormone and has multiple neuromodulatory functions in the brain. Its potent neuroprotective activities can be dependent and independent of estrogen receptors (ERs). In addition, E2 influences the processing of the amyloid beta precursor protein (APP), one central step in the pathogenesis of Alzheimer's disease. Here, we show: (a) that physiological concentrations of E2 very rapidly cause an increased release of secreted nonamyloidogenic APP (sAPPalpha) in mouse hippocampal HT22 and human neuroblastoma SK-N-MC cells; and (b) that this effect is mediated through E2 via the phosphorylation of extracellular-regulated kinase 1 and 2 (ERK1/2), prominent members of the mitogen-activated protein kinase (MAPK) pathway. Furthermore, we show that the activation of MAPK-signaling pathway and the enhancement of the sAPP release is independent of ERs and could be induced by E2 to a similar extent in neuronal cells either lacking or overexpressing a functional ER.  相似文献   

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