Fasting Drives Nrf2-Related Antioxidant Response in Skeletal Muscle.

A common metabolic condition for living organisms is starvation/fasting, a state that could play systemic-beneficial roles. Complex adaptive responses are activated during fasting to help the organism to maintain energy homeostasis and avoid nutrient stress. Metabolic rearrangements during fasting cause mild oxidative stress in skeletal muscle. The nuclear factor erythroid 2-related factor 2 (Nrf2) controls adaptive responses and remains the major regulator of quenching mechanisms underlying different types of stress. Here, we demonstrate a positive role of fasting as a protective mechanism against oxidative stress in skeletal muscle. In particular, by using in vivo and in vitro models of fasting, we found that typical Nrf2-dependent genes, including those controlling iron (e.g., Ho-1) and glutathione (GSH) metabolism (e.g., Gcl, Gsr) are induced along with increased levels of the glutathione peroxidase 4 (Gpx4), a GSH-dependent antioxidant enzyme. These events are associated with a significant reduction in malondialdehyde, a well-known by-product of lipid peroxidation. Our results suggest that fasting could be a valuable approach to boost the adaptive anti-oxidant responses in skeletal muscle.

APP Maturation and Intracellular Localization Are Controlled by a Specific Inhibitor of 37/67 kDa Laminin-1 Receptor in Neuronal Cells.

Amyloid precursor protein (APP) is processed along both the nonamyloidogenic pathway preventing amyloid beta peptide (Aß) production and the amyloidogenic pathway, generating Aß, whose accumulation characterizes Alzheimer's disease. Items of evidence report that the intracellular trafficking plays a key role in the generation of Aß and that the 37/67 kDa LR (laminin receptor), acting as a receptor for Aß, may mediate Aß-pathogenicity. Moreover, findings indicating interaction between the receptor and the key enzymes involved in the amyloidogenic pathway suggest a strong link between 37/67 kDa LR and APP processing. We show herein that the specific 37/67 kDa LR inhibitor, NSC48478, is able to reversibly affect the maturation of APP in a pH-dependent manner, resulting in the partial accumulation of the immature APP isoforms (unglycosylated/acetylated forms) in the endoplasmic reticulum (ER) and in transferrin-positive recycling endosomes, indicating alteration of the APP intracellular trafficking. These effects reveal NSC48478 inhibitor as a novel small molecule to be tested in disease conditions, mediated by the 37/67 kDa LR and accompanied by inactivation of ERK1/2 (extracellular signal-regulated kinases) signalling and activation of Akt (serine/threonine protein kinase) with consequent inhibition of GSK3ß.

miR-125b regulates the early steps of ESC differentiation through dies1 in a TGF-independent manner.

Over the past few years, it has become evident that the distinctive pattern of miRNA expression seen in embryonic stem cells (ESCs) contributes to important signals in the choice of the cell fate. Thus, the identification of miRNAs and their targets, whose expression is linked to a specific step of differentiation, as well as the modulation of these miRNAs, may prove useful in the learning of how ESC potential is regulated. In this context, we have studied the expression profile of miRNAs during neural differentiation of ESCs. We have found that miR-125b is upregulated in the first steps of neural differentiation of ESCs. This miRNA targets the BMP4 co-receptor, Dies1, and, in turn, regulates the balance between BMP4 and Nodal/Activin signaling. The ectopic expression of miR-125b blocks ESC differentiation at the epiblast stage, and this arrest is rescued by restoring the expression of Dies1. Finally, opposite to miR-125a, whose expression is under the control of the BMP4, miR-125b is not directly regulated by Transforming Growth Factor beta (TGFß) signals. These results highlight a new important role of miR-125b in the regulation of the transition from ESCs to the epiblast stage and add a new level of control on TGFß signaling in ESCs.

Fe65 matters: new light on an old molecule.

The discovery that the main constituents of amyloid deposits, characteristic of Alzheimer neuropathology, derive from the proteolytic processing of the membrane precursor amyloid precursor protein (APP) is one of the milestones of the research history of this disease. Despite years of intense studies, the functions of APP and of its amyloidogenic processing are still under debate. One focus of these studies was the complex network of protein-protein interactions centered at the cytosolic domain of APP, which suggests the involvement of APP in a lively signaling pathway. Fe65 was the first protein to be demonstrated to interact with the APP cytodomain. Starting from this observation, a large body of data has been gathered, indicating that Fe65 is an adaptor protein, which binds numerous proteins, further than APP. Among these proteins, the crosstalk with Mena, mDab, and Abl suggested the involvement of the Fe65-APP complex in the regulation of cell motility, with a relevant role in differentiation and development. Other partners, like the histone acetyltransferase Tip60, indicated the possibility that the nuclear fraction of Fe65 could be involved in gene regulation and/or DNA repair.

Nutritional limitation sensitizes mammalian cells to GSK-3ß inhibitors and leads to growth impairment.

The serine/threonine kinase GSK-3ß was initially described as a key enzyme involved in glucose metabolism, but it is now known to regulate a wide range of biological processes, including proliferation and apoptosis. We previously reported a transformation-dependent cell death induced by glucose limitation in K-ras-transformed NIH3T3. To address the mechanism of this phenomenon, we analyzed GSK-3ß regulation in these cells in conditions of high versus low glucose availability. We found that glucose depletion caused a marked inhibition of GSK-3ß through posttranslational mechanisms and that this inhibition was much less pronounced in normal cells. Further inhibition of GSK-3ß with lithium chloride, combined with glucose shortage, caused specific activation of AMP-activated protein kinase and significant suppression of proliferation in transformed but not normal cells. The cooperative effect of lithium and low glucose availability on cell growth did not seem to depend exclusively on ras pathway activation because two human cell lines, A549 and MDA-MB-231, both harboring an activated ras gene, showed very different sensitivity to lithium. These findings thus provide a rationale to further analyze the biochemical bases for combined glucose deprivation and GSK-3ß inhibition as a new approach to control transformed cell growth.

Fe65 is required for Tip60-directed histone H4 acetylation at DNA strand breaks.

Fe65 is a binding partner of the Alzheimer's beta-amyloid precursor protein APP. The possible involvement of this protein in the cellular response to DNA damage was suggested by the observation that Fe65 null mice are more sensitive to genotoxic stress than WT counterpart. Fe65 associated with chromatin under basal conditions and its involvement in DNA damage repair requires this association. A known partner of Fe65 is the histone acetyltransferase Tip60. Considering the crucial role of Tip60 in DNA repair, we explored the hypothesis that the phenotype of Fe65 null cells depended on its interaction with Tip60. We demonstrated that Fe65 knockdown impaired recruitment of Tip60-TRRAP complex to DNA double strand breaks and decreased histone H4 acetylation. Accordingly, the efficiency of DNA repair was decreased upon Fe65 suppression. To explore whether APP has a role in this mechanism, we analyzed a Fe65 mutant unable to bind to APP. This mutant failed to rescue the phenotypes of Fe65 null cells; furthermore, APP/APLP2 suppression results in the impairment of recruitment of Tip60-TRRAP complex to DNA double strand breaks, decreased histone H4 acetylation and repair efficiency. On these bases, we propose that Fe65 and its interaction with APP play an important role in the response to DNA damage by assisting the recruitment of Tip60-TRRAP to DNA damage sites.

MicroRNAs, Long Non-Coding RNAs, and Circular RNAs in the Redox Control of Cell Senescence.

Cell senescence is critical in diverse aspects of organism life. It is involved in tissue development and homeostasis, as well as in tumor suppression. Consequently, it is tightly integrated with basic physiological processes during life. On the other hand, senescence is gradually being considered as a major contributor of organismal aging and age-related diseases. Increased oxidative stress is one of the main risk factors for cellular damages, and thus a driver of senescence. In fact, there is an intimate link between cell senescence and response to different types of cellular stress. Oxidative stress occurs when the production of reactive oxygen species/reactive nitrogen species (ROS/RNS) is not adequately detoxified by the antioxidant defense systems. Non-coding RNAs are endogenous transcripts that govern gene regulatory networks, thus impacting both physiological and pathological events. Among these molecules, microRNAs, long non-coding RNAs, and more recently circular RNAs are considered crucial mediators of almost all cellular processes, including those implicated in oxidative stress responses. Here, we will describe recent data on the link between ROS/RNS-induced senescence and the current knowledge on the role of non-coding RNAs in the senescence program.

Coating of Flexible PDMS Substrates through Matrix-Assisted Pulsed Laser Evaporation (MAPLE) with a New-Concept Biocompatible Graphenic Material.

In this study, matrix-assisted pulsed laser evaporation (MAPLE) was used to deposit graphene-like materials (GL), a new class of biocompatible graphene-related materials (GRMs) obtained from a controlled top-down demolition of a carbon black, on silicone slices to test their potential use as functional coating on invasive medical devices as indwelling urinary catheters. Results indicate that the relevant chemical-physical features of the deposit (controlled by FTIR and AFM) were maintained after MAPLE deposition. After deposition, GL films underwent a biological survey toward target cellular lines (murine fibroblast NIH3T3, human keratinocytes HaCAT and the human cervical adenocarcinoma epithelial-like HeLa). Results indicate that the GL films did not lead to any perturbations in the different biological parameters evaluated. The presented results and the possibility to further functionalize the GL or combine them with other functional materials in a hybrid fashion to assure a tighter adhesion onto the substrate for use in harsh conditions open the door to practical applications of these new-concept medical devices (drug delivery, next generation flexible devices, multifunctional coatings) paving the way to the prevention of nosocomial infections driven by catheterization through antibiotics-free approaches.

miRNA 34a, 100, and 137 modulate differentiation of mouse embryonic stem cells.

MicroRNAs (miRNAs) play an important role in proper function and differentiation of mouse embryonic stem cells (ESCs). We performed a systematic comparison of miRNA expression in undifferentiated vs. differentiating ESCs. We report that 138 miRNAs are increased on the induction of differentiation. We compared the entire list of candidate mRNA targets of up-regulated miRNAs with that of mRNA down-regulated in ESCs on induction of differentiation. Among the candidate targets emerging from this analysis, we found three genes, Smarca5, Jarid1b, and Sirt1, previously demonstrated to be involved in sustaining the undifferentiated phenotype in ESCs. On this basis, we first demonstrated that Smarca5 is a direct target of miR-100, Jarid1b of miR-137, and we also confirmed previously published data demonstrating that Sirt1 is a direct target of miR-34a in a different context. The suppression of these three miRNAs by anti-miRs caused the block of ESC differentiation induced by LIF withdrawal. On the other hand, the overexpression of the three miRNAs resulted in an altered expression of differentiation markers. These results demonstrate that miR-34a, miR-100, and miR-137 are required for proper differentiation of mouse ESCs, and that they function in part by targeting Sirt1, Smarca5, and Jarid1b mRNAs.

Activation of amyloid precursor protein processing by growth factors is dependent on Ras GTPase activity.

The ß-amyloid peptide is generated by the proteolysis of the amyloid precursor protein (APP) by the action of ß- and γ-secretase. The mechanisms underlying this process are poorly understood. Using a cell-based reporter gene assay we analysed the possible signals and pathways that could be involved in APP cleavage. We used the stable cell line HeLa AG that expresses the human APP(695) fused with the yeast transcription factor Gal4. This fusion protein is normally translocated into the plasma membrane and after APP-Gal4 cleavage, the AICD-Gal4 fragment released can activate the transcription of a luciferase reporter gene. Through this reporter system, we demonstrated that Ras GTPase, but not Ral and Rap, could promote APP-Gal4 cleavage. In addition HeLa AG cells stimulated with EGF or PDGF or overexpressing EGFR exhibit increased APP proteolysis in a Ras-dependent way. This process is also dependent on γ-secretase activity, being abolished by the γ-secretase inhibitor DAPT.

Inhibition of 37/67kDa Laminin-1 Receptor Restores APP Maturation and Reduces Amyloid-ß in Human Skin Fibroblasts from Familial Alzheimer's Disease.

Alzheimer's disease (AD) is a fatal neurodegenerative disorder caused by protein misfolding and aggregation, affecting brain function and causing dementia. Amyloid beta (Aß), a peptide deriving from amyloid precursor protein (APP) cleavage by-and γ-secretases, is considered a pathological hallmark of AD. Our previous study, together with several lines of evidence, identified a strict link between APP, Aß and 37/67kDa laminin receptor (LR), finding the possibility to regulate intracellular APP localization and maturation through modulation of the receptor. Here, we report that in fibroblasts from familial AD (fAD), APP was prevalently expressed as an immature isoform and accumulated preferentially in the transferrin-positive recycling compartment rather than in the Golgi apparatus. Moreover, besides the altered mitochondrial network exhibited by fAD patient cells, the levels of pAkt and pGSK3 were reduced in respect to healthy control fibroblasts and were accompanied by an increased amount of secreted Aß in conditioned medium from cell cultures. Interestingly, these features were reversed by inhibition of 37/67kDa LR by NSC47924 a small molecule that was able to rescue the "typical" APP localization in the Golgi apparatus, with consequences on the Aß level and mitochondrial network. Altogether, these findings suggest that 37/67kDa LR modulation may represent a useful tool to control APP trafficking and Aß levels with implications in Alzheimer's disease.

A Functional Analysis of the Unclassified Pro2767Ser BRCA2 Variant Reveals Its Potential Pathogenicity that Acts by Hampering DNA Binding and Homology-Mediated DNA Repair.

BRCA1 and BRCA2 are the genes most frequently associated with hereditary breast and ovarian cancer (HBOC). They are crucial for the maintenance of genome stability, particularly in the homologous recombination-mediated repair pathway of DNA double-strand breaks (HR-DSBR). Widespread BRCA1/2 next-generation sequencing (NGS) screening has revealed numerous variants of uncertain significance. Assessing the clinical significance of these variants is challenging, particularly regarding the clinical management of patients. Here, we report the functional characterization of the unclassified BRCA2 c.8299C > T variant, identified in a young breast cancer patient during BRCA1/2 NGS screening. This variant causes the change of Proline 2767 to Serine in the DNA binding domain (DBD) of the BRCA2 protein, necessary for the loading of RAD51 on ssDNA during the HR-DSBR. Our in silico analysis and 3D-structure modeling predicted that the p.Pro2767Ser substitution is likely to alter the BRCA2 DBD structure and function. Therefore, to evaluate the functional impact of the p.Pro2767Ser variant, we used a minigene encoding a truncated protein that contains the BRCA2 DBD and the nearby nuclear localization sequence. We found that the ectopically expressed truncated protein carrying the normal DBD, which retains the DNA binding function and lacks the central RAD51 binding domain, interferes with endogenous wild-type BRCA2 mediator functions in the HR-DSBR. We also demonstrated that the BRCA2 Pro2767Ser DBD is unable to compete with endogenous BRCA2 DNA binding, thereby suggesting that the p.Pro2767Ser substitution in the full-length protein causes the functional loss of BRCA2. Consequently, our data suggest that the p.Pro2767Ser variant should be considered pathogenic, thus supporting a revision of the ClinVar interpretation. Moreover, our experimental strategy could be a valid method with which to preliminarily evaluate the pathogenicity of the unclassified BRCA2 germline variants in the DBD and their risk of predisposing to HBOC.

Adipocyte metabolism is improved by TNF receptor-targeting small RNAs identified from dried nuts.

There is a growing interest in therapeutically targeting the inflammatory response that underlies age-related chronic diseases including obesity and type 2 diabetes. Through integrative small RNA sequencing, we show the presence of conserved plant miR159a and miR156c in dried nuts having high complementarity with the mammalian TNF receptor superfamily member 1a (Tnfrsf1a) transcript. We detected both miR159a and miR156c in exosome-like nut nanovesicles (NVs) and demonstrated that such NVs reduce Tnfrsf1a protein and dampen TNF-α signaling pathway in adipocytes. Synthetic single-stranded microRNAs (ss-miRs) modified with 2'-O-methyl group function as miR mimics. In plants, this modification naturally occurs on nearly all small RNAs. 2'-O-methylated ss-miR mimics for miR156c and miR159a decreased Tnfrsf1a protein and inflammatory markers in hypertrophic as well as TNF-α-treated adipocytes and macrophages. miR156c and miR159a mimics effectively suppress inflammation in mice, highlighting a potential role of plant miR-based, single-stranded oligonucleotides in treating inflammatory-associated metabolic diseases.

Role of human GKN1 on APP processing in gastric cancer.

Gastrokine 1 (GKN1) is highly expressed in gastric tissue and is secreted into the stomach but is not expressed in gastric cancer. GKN1 belongs to the BRICHOS domain family and plays a major role in maintaining gastric mucosa integrity. We previously demonstrated that a recombinant human GKN1 protein was able to interact with the amyloid precursor protein (APP) and was endowed with an anti-amyloidogenic property because it inhibited polymerization of the Aß(1-40) peptide released from APP upon its partial hydrolysis. Here, we report that GKN1 can act as a physiological suppressor of Aß production in gastric cancer cells. GKN1 blocked the access of γ-secretase to APP, thereby facilitating the cleavage of APP by α- and ß-secretases. GKN1 directly interacted with APP C-terminal fragments, C83 and C99. In addition, it did not affect γ-secretase activity in gastric cancer cells because it did not alter Notch1 processing. GKN1-mediated inhibition of APP processing might represent a new approach for the prevention and therapy of Alzheimer's disease (AD).

The cytosolic domain of APP and its possible role in the pathogenesis of Alzheimer's disease.

APP is a type I membrane protein of unknown function, whose proteolytic processing, driven by beta- and gamma-secretases, generates the beta-amyloid peptides, one of the hallmarks of the pathogenesis of Alzheimer's disease. The short cytosolic domain of APP is the center of a complex network of protein-protein interactions. This network appears to play a crucial role in the regulation of the APP processing and in turn in the generation of the amyloid peptides, thus suggesting candidate targets for new therapeutic approaches. Furthermore, some possible functions of APP could just emerge from the study of this cytodomain and its partners.

An epigenomic role of Fe65 in the cellular response to DNA damage.

Previous findings describe Fe65 as a key protein in the cellular response to genotoxic stress. However, the precise molecular mechanism by which Fe65 contributes to DNA damage signaling remains unclear. In this study, we hypothesized that the transcriptional activity of Fe65 may contribute to DNA damage pathways by regulating gene expression patterns activated in response to genotoxic stress. To address this hypothesis, we mapped the global binding profile of Fe65 by chromatin immunoprecipitation (ChIP)-sequencing in the SK-N-SH cells exposed to genotoxic stress. Unexpectedly, the genome-wide location analysis showed a substantial enrichment of Fe65 in the promoter regions of coding genes linked to DNA damage signaling pathways. To further investigate the role of Fe65 in the transcriptional regulation of putative coding target genes identified by ChIP-seq, we performed microarray assays using wild-type (WT) or Fe65 deficient mouse embryonic fibroblasts (MEFs) exposed to oxidative stress with multiple recovery times. Gene ontology analysis of the Fe65-depedent transcriptome suggested that Fe65 modulates the expression of genes critical for DNA damage response. Motif enrichment analysis of regulatory regions occupied by Fe65 revealed a strong correlation with key transcription factors involved in DNA damage signaling pathways, including E2F1, p53, and Jun. Comparison of ChIP-sequencing results with microarray results ultimately identified 248 Fe65-depedent target genes, the majority of which were known regulators of cell cycle, cell death, and DNA replication and repair pathways. We validated the target genes identified by in silico analysis by qPCR experiments. Collectively, our results provide strong evidence that Fe65 plays a role in DNA damage response and cell viability by epigenomic regulation of specific transcriptional programs activated upon genotoxic stress.

Binding of carbonic anhydrase IX to 45S rDNA genes is prevented by exportin-1 in hypoxic cells.

Carbonic anhydrase IX (CA IX) is a surrogate marker of hypoxia, involved in survival and pH regulation in hypoxic cells. We have recently characterized its interactome, describing a set of proteins interacting with CA IX, mainly in hypoxic cells, including several members of the nucleocytoplasmic shuttling apparatuses. Accordingly, we described complex subcellular localization for this enzyme in human cells, as well as the redistribution of a carbonic anhydrase IX pool to nucleoli during hypoxia. Starting from this evidence, we analyzed the possible contribution of carbonic anhydrase IX to transcription of the 45 S rDNA genes, a process occurring in nucleoli. We highlighted the binding of carbonic anhydrase IX to nucleolar chromatin, which is regulated by oxygen levels. In fact, CA IX was found on 45 S rDNA gene promoters in normoxic cells and less represented on these sites, in hypoxic cells and in cells subjected to acetazolamide-induced acidosis. Both conditions were associated with increased representation of carbonic anhydrase IX/exportin-1 complexes in nucleoli. 45 S rRNA transcript levels were accordingly downrepresented. Inhibition of nuclear export by leptomycin B suggests a model in which exportin-1 acts as a decoy, in hypoxic cells, preventing carbonic anhydrase IX association with 45 S rDNA gene promoters.

Anti-amyloidogenic property of human gastrokine 1.

Gastrokine 1 (GKN1) is a stomach-specific protein expressed in normal gastric tissue but absent in gastric cancer. GKN1 plays a major role in maintaining gastric mucosa integrity and is characterized by the presence of a BRICHOS domain consisting of about 100 amino acids also found in several unrelated proteins associated with major human diseases like BRI2, related to familial British and Danish dementia and surfactant protein C (SP-C), associated with respiratory distress syndrome. It was reported that recombinant BRICHOS domains from BRI2 and SP-C precursor (proSP-C) prevent fibrils formation of amyloid-beta peptide (Aß), that is the major component of extracellular amyloid deposits in Alzheimer's disease. Here we investigated on the interaction between human recombinant GKN1 (rGKN1) and Aß peptide (1-40) that derives from the partial hydrolysis of the amyloid precursor protein (APP). GKN1 prevented amyloid aggregation and fibrils formation by inhibiting Aß(1-40) polymerization, as evaluated by SDS-PAGE, thioflavin-T binding assay and gel filtration experiments. Mass spectrometry showed the formation of a prevailing 1:1 complex between GKN1 and Aß(1-40). SPR analysis of GKN1/Aß interaction led to calculate a dissociation constant (KD) of 34 µM. Besides its interaction with Aß(1-40), GKN1 showed also to interact with APP as evaluated by confocal microscopy and Ni-NTA pull-down. Data strongly suggest that GKN1 has anti-amyloidogenic properties thus functioning as a chaperone directed against unfolded segments and with the ability to recognize amyloidogenic polypeptides and prevent their aggregation.

Notch activation induces neurite remodeling and functional modifications in SH-SY5Y neuronal cells.

Notch proteins are definitely recognized as key regulators of the neuronal fate during embryo development, but their function in the adult brain is still largely unknown. We have previously demonstrated that Notch pathway stimulation increases microtubules stability followed by the remodeling of neuronal morphology with neurite varicosities loss, thicker neuritis, and enlarged growth cones. Here we show that the neurite remodeling is a dynamic event, dependent on transcription and translation, and with functional implications. Exposure of differentiated human SH-SY5Y neuroblastoma cells to the Notch ligand Jagged1 induces varicosities loss all along the neurites, accompanied by the redistribution of presynaptic vesicles and the decrease in neurotransmitters release. As evaluated by time lapse digital imaging, dynamic changes in neurite morphology were rapidly reversible and dependent on the activation of the Notch signaling pathway. In fact, it was prevented by the inhibition of the proteolytic gamma-secretase enzyme or the transcription machinery, and was mimicked by the transfection of the intracellular domain of Notch. One hour after treatment with Jagged1, several genes were downregulated. Many of these genes encode proteins that are known to be involved in protein synthesis. These data suggest that in adult neurons, Notch pathway activates a transcriptional program that regulates the equilibrium between varicosities formation and varicosities loss in the neuronal presynaptic compartment involving the expression and redistribution of both structural and functional proteins.

Receptor- and non-receptor tyrosine kinases induce processing of the amyloid precursor protein: role of the low-density lipoprotein receptor-related protein.

The Alzheimer's beta-amyloid peptides derive from the proteolytic processing of the beta-amyloid precursor protein, APP, by beta- and gamma-secretases. The regulation of this processing is not fully understood. Experimental evidence suggests that the activation of pathways involving protein tyrosine kinases, such as PDGFR and Src, could induce the cleavage of APP and in turn the generation of amyloid peptides. In this paper we addressed the effect of receptor and nonreceptor protein tyrosine kinases on the cleavage of APP and the mechanisms of their action. To this aim, we developed an in vitro system based on the APP-Gal4 fusion protein stably transfected in SHSY5Y neuroblastoma cell line. The cleavage of this molecule, induced by various stimuli, results in the activation of the transcription of the luciferase gene under the control of Gal4 cis-elements. By using this experimental system we demonstrated that, similarly to Src, three tyrosine kinases, TrkA, Ret and EGFR, induced the cleavage of APP-Gal4. We excluded that this effect was mediated by the activation of Ras-MAPK, PI3K-Akt and PLC-gamma pathways. Furthermore, the direct phosphorylation of the APP cytosolic domain does not affect Abeta peptide generation. On the contrary, experiments in cells lacking the LDL-receptor related protein LRP support the hypothesis that the interaction of APP with LRP is required for the induction of APP cleavage by tyrosine kinases.