A ternary complex consisting of AICD,FE65, and TIP60 down-regulates Stathmin1

The ternary complex consisting of AICD/FE65/TIP60 is thought to play a role in gene expression and was suggested to have a crucial impact in Alzheimer's disease. AICD is the intracellular subdomain of the amyloid precursor protein (APP) and able to bind the adapter protein FE65 and the histone acetyltransferase TIP60 setting up a nuclear dot-like phenotype. Within this work we readdressed the generation of the complex as a function of its compartments. Subsequently, we studied the proteome of AFT expressing cells vs. controls and identified Stathmin1 significantly down-regulated in AFT cells. Stathmin1 functions as an important regulatory protein of microtubule dynamics and was found associated with neurofibrillary tangles in brains of Alzheimer's disease patients. We validated our results using an independent label-free mass spectrometry based method using the same cell culture model. In a reversal model with diminished APP expression, caused by simultaneous knock-down of all three members of the APP family, we further confirmed our results, as Stathmin1 was regulated in an opposite fashion. We hypothesize that AICD-dependent deregulation of Stathmin1 causes microtubule disorganization, which might play an important role for the pathophysiology of Alzheimer's disease.     

β-淀粉样蛋白前体蛋白胞内结构域（AICD）研究进展

老年性痴呆症（Alzheimer’s disease,AD）一个重要的病理学特征,是在神经细胞外形成由β-淀粉样蛋白（β-amyloid,Aβ）组成的淀粉样斑（amyloidplaques）。β-淀粉样蛋白前体蛋白（β-amyloidprocursorprotein,APP）经β-分泌酶和γ-分泌酶依次水解后产生AB和APP胞内结构域（APP intrace Uulardomain,AICD）。现在已经知道AB在AD的发病机制中起着关键作用,但是关于AICD的生理及病理功能还不清楚。近年来研究发现AICD可以与细胞内多种蛋白相互作用,而且AICD在基因转录、细胞凋亡以及APP的加工和运输过程中均有调节功能。本文针对这一领域的研究进展,对AICD的生理及病理功能进行探讨。     

Amyloid precursor protein intracellular domain modulates cellular calcium homeostasis and ATP content

Consecutive cleavages of amyloid precursor protein (APP) generate APP intracellular domain (AICD). Its cellular function is still unclear. In this study, we investigated the functional role of AICD in cellular Ca(2+) homeostasis. We could confirm previous observations that endoplasmic reticulum Ca(2+) stores contain less calcium in cells with reduced APP gamma-secretase cleavage products, increased AICD degradation, reduced AICD expression or in cells lacking APP. In addition, we observed an enhanced resting cytosolic calcium concentration under conditions where AICD is decreased or missing. In view of the reciprocal effects of Ca(2+) on mitochondria and of mitochondria on Ca(2+) homeostasis, we analysed further the cellular ATP content and the mitochondrial membrane potential. We observed a reduced ATP content and a mitochondrial hyperpolarisation in cells with reduced amounts of AICD. Blockade of mitochondrial oxidative phosphorylation chain in control cells lead to similar alterations as in cells lacking AICD. On the other hand, substrates of Complex II rescued the alteration in Ca(2+) homeostasis in cells lacking AICD. Based on these observations, our findings indicate that alterations observed in endoplasmic reticulum Ca(2+) storage in cells with reduced amounts of AICD are reciprocally linked to mitochondrial bioenergetic function.     

Structure of the intracellular domain of the amyloid precursor protein in complex with Fe65-PTB2

Cleavage of the amyloid precursor protein (APP) is a crucial event in Alzheimer disease pathogenesis that creates the amyloid-beta peptide (Abeta) and liberates the carboxy-terminal APP intracellular domain (AICD) into the cytosol. The interaction of the APP C terminus with the adaptor protein Fe65 mediates APP trafficking and signalling, and is thought to regulate APP processing and Abeta generation. We determined the crystal structure of the AICD in complex with the C-terminal phosphotyrosine-binding (PTB) domain of Fe65. The unique interface involves the NPxY PTB-binding motif and two alpha helices. The amino-terminal helix of the AICD is capped by threonine T(668), an Alzheimer disease-relevant phosphorylation site involved in Fe65-binding regulation. The structure together with mutational studies, isothermal titration calorimetry and nuclear magnetic resonance experiments sets the stage for understanding T(668) phosphorylation-dependent complex regulation at a molecular level. A molecular switch model is proposed.     

Amyloid precursor protein promotes endoplasmic reticulum stress-induced cell death via C/EBP homologous protein-mediated pathway

The accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER) is known to activate the ER, which is termed ER stress. Here, we demonstrated that amyloid precursor protein (APP) is a novel mediator of ER stress-induced apoptosis through the C/EBP homologous protein (CHOP) pathway. Expression of APP mRNA was elevated by tunicamycin- or dithiothreitol-induced ER stress. The levels of C83 and APP intracellular domain (AICD) fragments, which are cleaved from APP, were significantly increased under ER stress, although the protein level of full-length APP was decreased. Cellular viability was reduced in APP-over-expressing cells, which was attenuated by treatment with a γ-secretase inhibitor, N -[ N -(3,5-difluorophenacetyl)-L-alanyl]- S -phenylglycine t -butyl ester (DAPT). Cellular viability was also reduced in AICD-FLAG-over-expressing cells. The mRNA and protein levels of CHOP, an ER stress-responsive gene, were remarkably increased by APP over-expression, which was attenuated by treatment with DAPT. CHOP mRNA induction was also found in AICD-FLAG-over-expressing cells. Cell death and CHOP up-regulation by ER stress were attenuated by APP knockdown. Data obtained with a luciferase assay and chromatin immunoprecipitation assay indicated that AICD associates with the promoter region of the CHOP gene. In conclusion, ER stress-induced APP undergoes α- and γ-secretase cleavage and subsequently induces CHOP-mediated cell death.     

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.     

The intracellular domain of amyloid precursor protein interacts with FKBP12

To elucidate the roles of the APP intracellular domain (AICD) in the development of Alzheimer's disease, a yeast two-hybrid system was used to screen for AICD-interacting proteins. Our result revealed that FKBP12, an immunophilin with a peptidyl-prolyl cis-trans isomerase (PPIase) activity, may interact with AICD. This interaction was confirmed by coimmunoprecipitation studies. FKBP12 has been shown to be expressed at a higher level in areas of pathology of patients with neurodegenerative diseases. In addition, Pin1, a member of another PPIase family, has been suggested to be involved in the amyloidogenic APP processing and Abeta production. The interaction between FKBP12 and AICD might hint at a possible role FKBP12 plays, probably in a fashion similar to Pin1, in the amyloidogenesis of APP. We also found that the interaction was interfered, in a dose-dependent manner, by FK506, whose neuroprotective effect has been suggested to be correlated with its PPIase inhibitory activity.     

Amyloid precursor protein regulates migration and metalloproteinase gene expression in prostate cancer cells

Amyloid precursor protein (APP) is a type I transmembrane protein, and one of its processed forms, β-amyloid, is considered to play a central role in the development of Alzheimer’s disease. We previously showed that APP is a primary androgen-responsive gene in prostate cancer and that its increased expression is correlated with poor prognosis for patients with prostate cancer. APP has also been implicated in several human malignancies. Nevertheless, the mechanism underlying the pro-proliferative effects of APP on cancers is still not well-understood. In the present study, we explored a pathophysiological role for APP in prostate cancer cells using siRNA targeting APP (siAPP). The proliferation and migration of LNCaP and DU145 prostate cancer cells were significantly suppressed by siAPP. Differentially expressed genes in siAPP-treated cells compared to control siRNA-treated cells were identified by microarray analysis. Notably, several metalloproteinase genes, such as ADAM10 and ADAM17, and epithelial–mesenchymal transition (EMT)-related genes, such as VIM, and SNAI2, were downregulated in siAPP-treated cells as compared to control cells. The expression of these genes was upregulated in LNCaP cells stably expressing APP when compared with control cells. APP-overexpressing LNCaP cells exhibited enhanced migration in comparison to control cells. These results suggest that APP may contribute to the proliferation and migration of prostate cancer cells by modulating the expression of metalloproteinase and EMT-related genes.     

Changes of neuron-specific and apoptosis gene expression levels after ventromedial hypothalamic lesions in rat intestine

The intestinal epithelium is continuously renewed through a balance between cell proliferation and apoptosis. We identified genes of which expression profiles showed significant modulation, and we investigated the cellular mechanisms of this gene regulation in rat intestine after ventromedial hypothalamic (VMH) lesions. Total RNA was extracted, and differences in the gene expression profiles between rats at day 3 after VMH lesioning and in sham-VMH lesioned rats were investigated using DNA microarray analysis and real-time polymerase chain reaction (PCR) methods. DNA microarray analysis revealed that VMH lesions regulated the genes that were involved in functions predominantly related to neuronal development, cell proliferation and apoptosis. Real-time PCR also confirmed that gene expressions of Efnb1 were downregulated. Meanwhile, expression of Casp3 was similar. It is noted that the signaling networks of many gene families, including neuron-specific genes and apoptosis genes in the intestine were changed after VMH lesioning. VMH lesions may suppress mainly the caspase independent type II pathway for apoptosis and induce cell proliferation in the intestine.     

Regulated intramembrane proteolysis of amyloid precursor protein and regulation of expression of putative target genes

gamma-Secretase-dependent regulated intramembrane proteolysis of amyloid precursor protein (APP) releases the APP intracellular domain (AICD). The question of whether this domain, like the Notch intracellular domain, is involved in nuclear signalling is highly controversial. Although some reports suggest that AICD regulates the expression of KAI1, glycogen synthase kinase-3beta, Neprilysin and APP, we found no consistent effects of gamma-secretase inhibitors or of genetic deficiencies in the gamma-secretase complex or the APP family on the expression levels of these genes in cells and tissues. Finally, we demonstrate that Fe65, an important AICD-binding protein, transactivates a wide variety of different promoters, including the viral simian virus 40 promoter, independent of AICD coexpression. Overall, the four currently proposed target genes are at best indirectly and weakly influenced by APP processing. Therefore, inhibition of APP processing to decrease Abeta generation in Alzheimer's disease will not interfere significantly with the function of these genes.