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Neurogenesis impairment starting from early developmental stages is a key determinant of intellectual disability in Down syndrome (DS). Previous evidence provided a causal relationship between neurogenesis impairment and malfunctioning of the mitogenic Sonic Hedgehog (Shh) pathway. In particular, excessive levels of AICD (amyloid precursor protein intracellular domain), a cleavage product of the trisomic gene APP (amyloid precursor protein) up-regulate transcription of Ptch1 (Patched1), the Shh receptor that keeps the pathway repressed. Since AICD results from APP cleavage by γ-secretase, the goal of the current study was to establish whether treatment with a γ-secretase inhibitor normalizes AICD levels and restores neurogenesis in trisomic neural precursor cells. We found that treatment with a selective γ-secretase inhibitor (ELND006; ELN) restores proliferation in neurospheres derived from the subventricular zone (SVZ) of the Ts65Dn mouse model of DS. This effect was accompanied by reduction of AICD and Ptch1 levels and was prevented by inhibition of the Shh pathway with cyclopamine. Treatment of Ts65Dn mice with ELN in the postnatal period P3–P15 restored neurogenesis in the SVZ and hippocampus, hippocampal granule cell number and synapse development, indicating a positive impact of treatment on brain development. In addition, in the hippocampus of treated Ts65Dn mice there was a reduction in the expression levels of various genes that are transcriptionally regulated by AICD, including APP, its origin substrate. Inhibitors of γ-secretase are currently envisaged as tools for the cure of Alzheimer's disease because they lower βamyloid levels. Current results provide novel evidence that γ-secretase inhibitors may represent a strategy for the rescue of neurogenesis defects in DS. [ABSTRACT FROM AUTHOR]

Abstract: Intellectual disability in Down syndrome (DS) appears to be related to severe neurogenesis impairment during brain development. The molecular mechanisms underlying this defect are still largely unknown. Accumulating evidence has highlighted the importance of GSK3β signaling for neuronal precursor proliferation/differentiation. In neural precursor cells (NPCs) from Ts65Dn mice and human fetuses with DS, we found reduced GSK3β phosphorylation and, hence, increased GSK3β activity. In cultures of trisomic subventricular-zone-derived adult NPCs (aNPCs) we found that deregulation of GSK3β activity was due to higher levels of the AICD fragment of the trisomic gene APP that directly bound to GSK3β. We restored GSK3β phosphorylation in trisomic aNPCs using either lithium, a well-known GSK3β inhibitor, or using a 5-HT receptor agonist or fluoxetine, which activated the serotonin receptor 5-HT1A. Importantly, this effect was accompanied by restoration of proliferation, cell fate specification and neuronal maturation. In agreement with results obtained in vitro, we found that early treatment with fluoxetine, which was previously shown to rescue neurogenesis and behavior in Ts65Dn mice, restored GSK3β phosphorylation. These results provide a link between GSK3β activity alteration, APP triplication and the defective neuronal production that characterizes the DS brain. Knowledge of the molecular mechanisms underlying neurogenesis alterations in DS may help to devise therapeutic strategies, potentially usable in humans. Results suggest that drugs that increase GSK3β phosphorylation, such as lithium or fluoxetine, may represent useful tools for the improvement of neurogenesis in DS. [Copyright &y& Elsevier]

Abstract: Amyloid precursor protein (APP), a key protein in pathogenesis of Alzheimer’s disease (AD), is a type I transmembrane protein which can be cleaved by β- and γ-secretase to release the amyloidogenic β-amyloid peptides (Aβ) and the APP intracellular domain (AICD). While Aβ has been widely believed to initiate pathogenic cascades culminating AD, the physiological functions and regulations of AICD remain elusive. In present study, endogenous AICD was demonstrated to be increased by canonical Wnt signal. Instead of due to γ-secretase activity, enhanced AICD expression was found due to the increased protein stability by Wnt/β-catenin. β-Catenin was demonstrated to be an associating partner of AICD, capable of promoting AICD mediated transcriptional activity. Investigation by AICD mutants proved that Fe65, a previously identified AICD binding partner, is not involved in this regulation. Taken together, our results suggest that AICD is stabilized and the AICD mediated transcriptional activity is promoted by canonical Wnt/β-catenin signaling independent of Fe65. [Copyright &y& Elsevier]

Abstract: Alzheimer''s disease (AD) is a neurodegenerative disorder that represents the most common type of dementia in the elderly. One of the hallmarks of this disease is a progressive accumulation of amyloid fibrils in senile plaques (SPs), which are composed principally of amyloid-β peptides (Aβ). The 4-kDa β-amyloid peptides are produced from the β-amyloid precursor protein (APP) through sequential processing by β- and γ-secretase enzymes in the amyloidogenic pathway. By an alternative non-amyloidogenic pathway, mediated by α- and γ-secretases enzymes, APP is processed within the Aβ domain. Both processing pathways may result in the generation of a fragment called APP intracellular C-terminal domain (AICD) which is hypothesized to contribute to the pathophysiology of AD. Experimental evidence highlights that biological functions of AICD are mediated by interactions between its YENPTY motif and specific binding factors. We critically reviewed literature concerning physiological function of this proteolitic fragment, mainly focusing on their degradation by the two best characterized systems, proteasome and IDE (insulin degrading enzyme). Our work is aimed to analyse the functional role of AICD, integrating also the AICD degradation processes, to better define a potential role of AICD in signal transduction. [Copyright &y& Elsevier]

Abstract: Although there is compelling evidence that the β amyloid peptide (Aβ) can be centrally involved in Alzheimer''s disease, the natural role (if any) of this peptide remains unclear. Here we use green fluorescent protein (GFP) fusions to demonstrate that the Aβ sequence, like prion domains, can act as a modular aggregation domain when terminally appended to a normally soluble protein. We find that a single amino acid substitution (Leu17 to Pro) in the β peptide sequence can abolish this cis capacity to induce aggregation. Introduction of this substitution into full-length APP (i.e., a Leu613Pro substitution in APP695) alters the processing of APP leading to the accumulation of the C99 C-terminal fragment (CTF). We suggest that in at least some aggregation disease-related proteins the presence of an aggregation domain is not “accidental”, but reflects a selected role of these domains in modulating the trafficking or metabolism of the parental protein. [Copyright &y& Elsevier]

Abstract: A sequence of amyloid precursor protein (APP) cleavages culminates in the sequential release of the APP intracellular domain (AICD) and the amyloid β peptide (Aβ) and/or p3 fragment. One of the environmental factors favouring the accumulation of AICD appears to be a rise in intracellular pH. Here we further identified the metabolism and subcellular localization of artificially expressed constructs under such conditions. We also co-examined the mechanistic lead up to the AICD accumulation and explored possible significances for its increased expression. We found that most of the AICD generated under pH neutralized conditions is likely cleaved from C83. While the AICD surplus was unable to further activate transcription of a luciferase reporter via a Gal4-DNA-binding domain, it failed entirely via the endogenous promoter regions of proposed target genes, APP and KAI1. The lack of a specific transactivation potential was also demonstrated by the unchanged levels of target gene mRNA. However, rather than translocating to the nucleus, the AICD surplus remains membrane tethered or free in the cytosol where it interacts with Fe65. Therefore we provide strong evidence that an increase in AICD generation does not directly promote gene activation of previously proposed target genes. [Copyright &y& Elsevier]

Abstract: The processing of amyloid precursor protein (APP) by γ-secretase generates the APP intracellular domain (AICD), which functions as a transcriptional factor for target gene activation following localization into the nucleus. In this study, we demonstrate that AICD could be modified via covalent conjugation with Nedd8, a ubiquitin-like protein. Domain analysis and site-directed substitution of neddylation sites showed that multiple lysine residues of the APP C-terminal C99 fragment including AICD were acceptor sequences for Nedd8 conjugation. AICD-mediated transcriptional activation was inhibited by Nedd8 conjugation. Furthermore, the transcriptional activity of the neddylation-defective AICD mutant was not altered by Nedd8 expression. Nedd8 conjugation of AICD inhibited its interaction with Fe65, and consequently resulted in the impairment of AICD–Fe65–Tip60 complex formation for the transcriptional activation of the target gene. These results illustrate the regulatory mechanisms by which AICD transcriptional activity might be regulated via covalent conjugation with Nedd8. [Copyright &y& Elsevier]

Abstract: The APP intracellular domain (AICD) could be involved in signaling via interaction with the adaptor protein Fe65, and with the histone acetyl transferase Tip60. However, the real function of AICD and Fe65 in regulation of transcription remains controversial. In this study, the human APPGal4 fusion protein was expressed in CHO cells and the transcriptional activity of AICDGal4 was measured in a luciferase-based reporter assay. AICDGal4 was stabilized by expression of Fe65 and levels of AICDGal4 controlled luciferase activity. On the contrary, when human APP was expressed in CHO cells, coexpression of Fe65 increased luciferase activity without affecting the amount of AICD fragment. AICD produced from APP was protected from degradation by orthophenanthroline, but not by lactacystine, indicating that AICD is not a substrate of the chymotryptic activity of the proteasome. It is concluded that Fe65 can control luciferase activity without stabilizing the labile AICD fragment. [Copyright &y& Elsevier]

Abstract: Activation-induced cell death (AICD) plays an important role in maintenance of peripheral lymphocyte homeostasis. Reactive oxygen species (ROS) combined with simultaneous calcium (Ca2+) influx into the cytosol are required for induction of AICD. The extract obtained from the stem bark of Mangifera indica L. has shown to protect T cells from in vitro AICD. This extract is rich in polyphenolic compounds, the three main components of which are mangiferin (MA), catechin (C) and epicatechin (EC). The present study has focused on the possible contribution of the polyphenols MA, C and EC to the demonstrated protective effect of M. indica extract on in vitro human T cell AICD. Our results show that these polyphenols diminished the increase of intracellular ROS and free Ca2+ induced by T cell receptor (TCR) triggering. In addition, these polyphenols attenuated AICD. Our findings suggest that the T cell survival effect of M. indica extract is mediated, at least in part, by its main polyphenols. [Copyright &y& Elsevier]

Abstract: Amyloid precursor protein (APP)-derived intracellular domain (AICD) has a cytotoxic effect on neuronal cells and also participates in the regulation of gene transactivation. However, the precise molecular mechanisms behind the AICD-mediated apoptosis remain unknown. In this study, we have demonstrated that AICD interacts with p53 and enhances its transcriptional and pro-apoptotic functions. p53 was induced to be accumulated and associated with APP in response to cisplatin. Indeed, APP-C57 was co-immunoprecipitated with the endogenous p53. Enforced expression of APP-C57 or APP-C59 in U2OS cells bearing wild-type p53 led to an increase in number of apoptotic cells, whereas they had undetectable effects on p53-deficient H1299 cells, suggesting that AICD contributes to the activation of the p53-mediated apoptotic pathway. Consistent with this notion, the p53-mediated transcriptional activation and apoptosis were significantly enhanced by co-expression with APP-C57 or APP-C59. Thus, our present results strongly suggest that AICD triggers apoptosis through the p53-dependent mechanisms. [Copyright &y& Elsevier]

Abstract: The accumulation and deposition of fibrillar Aβ is thought to be the primary cause of Alzheimer’s disease. Aβ is derived from Alzheimer amyloid precursor protein (APP) by sequential proteolytic cleavage involving β- and γ-secretase. Recently, γ-secretase was shown to cleave near the cytoplasmic membrane boundary of APP (called the ε-cleavage), as well as in the middle of the membrane domain (γ-cleavage). It has been reported that the C-terminus of Aβ is generated via a series of sequential cleavages, ε-cleavage followed by γ-cleavage. However, recent article has reported that γ- and ε-site cleavage are regulated independently. The relationship between γ-site and ε-site cleavage is still unknown. In this study, we analyzed the generation of AICD and Aβ in CHO cells expressing APP derivatives. We found that ε-site cleavage preferentially occurs α-secretase processing product, and that Aβ 11–40/42 was generated without γ-secretase ε-site cleavage, indicating that γ-site cleavage and ε-site cleavage were regulated differentially. [Copyright &y& Elsevier]

Understanding the molecular mechanism of β-amyloid (Aβ) generation is crucial for Alzheimer''s disease pathogenesis as well as for normal APP function. The transmembrane domain (TM) of APP appears to undergo presenilin-dependent γ-secretase cleavage at two topologically distinct sites: a site in the middle of the TM domain that is crucial for the generation of Aβ-peptides, and a site close to the cytoplasmic border (S3-like/ɛ site) of the TM domain that leads to production of the APP intracellular domain (CTFγ/AICD). We demonstrate that, in contrast to the unique effect of familial Alzheimer''s disease (FAD) mutations in APP on Aβ42 production, some but not all FAD mutations also affect CTFγ generation. Furthermore, changes in total CTFγ levels do not correlate with either an increase or a decrease of any Aβ species, and inhibition of Aβ-peptide formation starting from position +1 (Aβ1–x) does not affect CTFγ production. These results suggest that cleavage at the γ40/42- and the S3-like sites can be dissociated, and that APP signaling and Aβ production are not tightly linked. [Copyright &y& Elsevier]

Cholesterol is one of multiple factors, other than familial genetic mutations, that can influence amyloid-β peptide (Aβ) metabolism and accumulation in Alzheimer disease (AD). The effect of a high-cholesterol diet on amyloid precursor protein (APP) processing in brain has not been thoroughly studied. This study was designed to further investigate the role of cholesterol in the production of Aβ and APP intracellular domain (AICD) in 12-month-old Tg2576 transgenic mice. The mice were maintained on a high-cholesterol diet for 6 weeks. We found that diet-induced hypercholesterolemia increased the APP cytosolic fragment AICD and reduced sAPPα in the Tg2576 mice compared to the mice on a control basal diet. In addition, the levels of detergent-extracted Aβ40 were reduced, although no change in guanidine-extracted Aβ levels was observed. Full-length APP, α/βC-terminal fragment (α/βCTF), and β-secretase (BACE) were not different in the cholesterol-fed mice compared to the control diet-fed mice. This study suggests that a high dietary cholesterol in aged mice may not only influence Aβ metabolism, but also regulate the AICD levels. AICD has a proposed role in signal transduction and apoptosis, hence modulation of AICD production could be an alternative mechanism by which cholesterol contributes to AD pathogenesis. [Copyright &y& Elsevier]

One of the cardinal neuropathological findings in brains from Alzheimer’s disease (AD) patients is the occurrence of amyloid β-peptide (Aβ) deposits. The γ-secretase-mediated intramembrane proteolysis event generating Aβ also results in the release of the APP intracellular domain (AICD), which may mediate nuclear signaling. It was recently shown that AICD starts at a position distal to the site predicted from γ-secretase cleavage within the membrane. This novel site, the ϵ site, is located close to the inner leaflet of the membrane bilayer. The relationship between proteolysis at the γ and ϵ sites has not been fully characterized. Here we studied AICD signaling in intact cells using a chimeric C99 molecule and a luciferase reporter system. We show that the release of AICD from the membrane takes place in a compartment downstream of the endoplasmic reticulum, is dependent on presenilin proteins, and can be inhibited by treatment with established γ-secretase inhibitors. Moreover, we find that AICD signaling remains unaltered from C99 derivatives containing mutations associated with increased Aβ42 production and familial AD. These findings indicate that there are very similar routes for Aβ and AICD formation but that FAD-linked mutations in APP primarily affect γ-secretase-mediated Aβ42 formation, and not AICD signaling. [Copyright &y& Elsevier]

Activation-induced cell death (AICD) represents a major means of peripheral tolerance induction, eliminating effector cells. NOD mice, a widely used model for autoimmune diabetes, are characterized by high levels of circulating T lymphocytes and by resistance to several apoptosis-inducing signals. The aim of this study was to analyse AICD in peripheral NOD T lymphocytes. First, we demonstrated in an in vitro AICD model that NOD T lymphocytes are more resistant to AICD (64±2%) compared to non-autoimmune C57BL/6 T lymphocytes (73±2%), but also diabetes-resistant NOR T lymphocytes (76±3%, P<0.05). Moreover, both CD4+and CD8+subsets were affected. Analysis of the cellular and molecular pathways revealed lower caspase 8 levels, a central caspase proximally involved in the AICD-pathway (fluorescence of 258±47 in NOD vs. 441±16 in NOR and 414±61 in C57BL/6 T lymphocytes, P<0.05). Gene expression analysis using real-time RT-PCR additionally revealed low expression of Fas and FasL, the death receptor system activating caspase 8 and contributing to AICD. Additionally, low IL-2 levels, together with high TGFβ and Bclx-L levels, confirm the presence of a NOD-specific AICD-resistance profile. In conclusion, we present cellular and molecular evidence for disturbed AICD mechanisms in NOD T lymphocytes. This resistance in AICD may contribute to defective tolerance induction to autoantigens in NOD mice. [Copyright &y& Elsevier]

Lung cancer remains the leading cause of cancer-related deaths and despite extensive research, the survival rate of lung cancer patients remains significantly low. Recent data reveal that aberrant Kras signaling drives regulatory T cells (Tregs) present in lung tumor microenvironment to establish immune deregulation and immunosuppression but the exact pathogenic mechanism is still unknown. In this study, we investigate the role of oncogenic Kras in Treg-related immunosuppression and its involvement in tumor-associated metabolic reprogramming. Findings reveal Tregs to prompt GATA3/NOS2-related immunosuppression via STING inhibition which triggers a decline in CD4+ T infiltration, and a subsequent increase in lung metastatic burden. Enhanced Treg expression was also associated with low T/MDSC ratio through restriction of CD8+CD44+CD62L− T effector cells, contributing to a tumor-promoting status. Specifically, TIM3+/LAG3+ Tregs prompted Kras-related immunosuppressive chemoresistance and were associated with T cell dysfunction. This Treg-dependent immunosuppression correlated with CD8 T cell exhaustion phenotype and ILC2 augmentation in mice. Moreover, enhanced Treg expression promoted activation-induced cell death (AICD) of T lymphocytes and guided lymph node metastasis in vivo. Overall, these findings demonstrate the multifaceted roles of Tregs in sustaining lung immunosuppressive neoplasia through tumor microenvironment remodeling and provide new opportunities for effective metastasis inhibition, especially in chemoresistant tumors. Unlabelled Image • Tregs trigger GATA3/NOS2-related immunosuppression via STING inhibition. • Enhanced Treg expression was associated with low T/MDSC ratio and CD8 + CD44 + CD62L- restriction. • TIM3+/LAG3+ Tregs prompted Kras-related immunosuppressive chemoresistance. • Treg expression promoted activation-induced cell death (AICD) of T lymphocytes. • Aberrant Treg signaling triggers metabolic reprogramming of metastatic lung tumor cells. [ABSTRACT FROM AUTHOR]