Emanuela Repetto

Amyloid precursor protein and presenilin1 interact with the adaptor GRB2 and modulate ERK1,2 signaling

The amyloid precursor protein (APP) and the presenilins 1 and 2 are genetically linked to the development of familial Alzheimer disease. APP is a single-pass transmembrane protein and precursor of fibrillar and toxic amyloid-β peptides, which are considered responsible for Alzheimer disease neurodegeneration. Presenilins are multipass membrane proteins, involved in the enzymatic cleavage of APP and other signaling receptors and transducers. The role of APP and presenilins in Alzheimer disease development seems to be related to the formation of amyloid-β peptides; however, their physiological function, reciprocal interaction, and molecular mechanisms leading to neurodegeneration are unclear. APP and presenilins are also involved in multiple interactions with intracellular proteins, the significance of which is under investigation. Among the different APP-interacting proteins, we focused our interest on the GRB2 adaptor protein, which connects cell surface receptors to intracellular signaling pathways. In this study we provide evidence by co-immunoprecipitation experiments, confocal and electron microscopy, and by fluorescence resonance energy transfer experiments that both APP and presenilin1 interact with GRB2 in vesicular structures at the centrosome of the cell. The final target for these interactions is ERK1,2, which is activated in mitotic centrosomes in a PS1- and APP-dependent manner. These data suggest that both APP and presenilin1 can be part of a common signaling pathway that regulates ERK1,2 and the cell cycle.

Low-density lipoprotein receptor-related protein promotes amyloid precursor protein trafficking to lipid rafts in the endocytic pathway

The major defining pathological hallmark of Alzheimers disease (AD) is the accumulation of amyloid β protein (Aβ), a small peptide derived from β‐ and γ‐secretase cleavages of the amyloid precursor protein (APP). Recent studies have shown that β‐ and γ‐secretase activities of BACE1 and prese‐nilin, respectively, are concentrated in intracellular lipid raft microdomains. However, the manner in which APP normally traffics to lipid rafts is unknown. In this study, using transient transfection and immuno‐precip‐itation assays, we show that the cytoplasmic domain of low‐density lipoprotein receptor‐related protein (LRP) interacts with APP and increases Aβ secretion and APP β‐CTF (C‐terminal fragment) generation by promoting BACE1‐APP interaction. We also employed discontinuous sucrose density gradient ultracentrifugation to show that the LRP cytoplasmic domain‐mediated effect was accompanied by greatly increased localization of APP and BACE1 to lipid raft membranes, where β‐ and γ‐secretase activities are highly enriched. Moreover, we provide evidence that endogenous LRP is required for the normal delivery of APP to lipid rafts and Aβ generation primarily in the endocytic but not secretory pathway. These results may provide novel insights to block Aβ generation by targeting LRP‐mediated delivery of APP to raft microdomains.—Yoon I.‐S., Chen, E., Busse, T., Repetto, E., Lakshmana, M. K., Koo, E. H., Kang D. E. Low‐density lipoprotein receptor‐related protein promotes amyloid precursor protein trafficking to lipid rafts in the endocytic pathway. FASEB J. 21, 2742–2752 (2007)

Presenilin 1 Regulates Epidermal Growth Factor Receptor Turnover and Signaling in the Endosomal-Lysosomal Pathway

Mutations in the gene encoding presenilin 1 (PS1) cause the most aggressive form of early-onset familial Alzheimer disease. In addition to its well established role in Aβ production and Notch proteolysis, PS1 has been shown to mediate other physiological activities, such as regulation of the Wnt/β-catenin signaling pathway, modulation of phosphatidylinositol 3-kinase/Akt and MEK/ERK signaling, and trafficking of select membrane proteins and/or intracellular vesicles. In this study, we present evidence that PS1 is a critical regulator of a key signaling receptor tyrosine kinase, epidermal growth factor receptor (EGFR). Specifically, EGFR levels were robustly increased in fibroblasts deficient in both PS1 and PS2 (PS-/-) due to delayed turnover of EGFR protein. Stable transfection of wild-type PS1 but not PS2 corrected EGFR to levels comparable to PS+/+ cells, while FAD PS1 mutations showed partial loss of activity. The C-terminal fragment of PS1 was sufficient to fully reduce EGFR levels. In addition, the rapid ligand-induced degradation of EGFR was markedly delayed in PS-/- cells, resulting in prolonged signal activation. Despite the defective turnover of EGFR, ligand-induced autophosphorylation, ubiquitination, and endocytosis of EGFR were not affected by the lack of PS1. Instead, the trafficking of EGFR from early endosomes to lysosomes was severely delayed by PS1 deficiency. Elevation of EGFR was also seen in brains of adult mice conditionally ablated in PS1 and in skin tumors associated with the loss of PS1. These findings demonstrate a critical role of PS1 in the trafficking and turnover of EGFR and suggest potential pathogenic effects of elevated EGFR as well as perturbed endosomal-lysosomal trafficking in cell cycle control and Alzheimer disease.

Immunohistochemical localization and biological activity of the steroidogenic enzyme cytochrome P450 17α-hydroxylase/C17, 20-lyase (P450C17) in the frog brain and pituitary

It is now clearly established that the brain has the capability of synthesizing various biologically active steroids including 17‐hydroxypregnenolone (17OH‐Δ5P), 17‐hydroxyprogesterone (17OH‐P), dehydroepiandrosterone (DHEA) and androstenedione (Δ4). However, the presence, distribution and activity of cytochrome P450 17α‐hydroxylase/C17, 20‐lyase (P450C17), a key enzyme required for the conversion of pregnenolone (Δ5P) and progesterone (P) into these steroids, are poorly documented. Here, we show that P450C17‐like immunoreactivity is widely distributed in the frog brain and pituitary. Prominent populations of P450C17‐containing cells were observed in a number nuclei of the telencephalon, diencephalon, mesencephalon and metencephalon, as well as in the pars distalis and pars intermedia of the pituitary. In the brain, P450C17‐like immunoreactivity was almost exclusively located in neurons. In several hypothalamic nuclei, P450C17‐positive cell bodies also contained 3β‐hydroxysteroid dehydrogenase‐like immunoreactivity. Incubation of telencephalon, diencephalon, mesencephalon, metencephalon or pituitary explants with [3H]Δ5P resulted in the formation of several tritiated steroids including 17OH‐Δ5P, 17OH‐P, DHEA and Δ4. De novo synthesis of C21 17‐hydroxysteroids and C19 ketosteroids was reduced in a concentration‐dependent manner by ketoconazole, a P450C17 inhibitor. This is the first detailed immunohistochemical mapping of P450C17 in the brain and pituitary of any vertebrate. Altogether, the present data provide evidence that CNS neurons and pituitary cells can synthesize androgens.

Apoptotic cell death influences the signaling activity of the amyloid precursor protein through ShcA and Grb2 adaptor proteins in neuroblastoma SH‐SY5Y cells

The amyloid precursor protein (APP) is an ubiquitous receptor‐like molecule involved in the pathogenesis of Alzheimers disease (AD). APP and some of its C‐terminal proteolytic fragments (CTFs) have been shown to be phosphorylated and to interact with cytosolic phosphotyrosine binding (PTB) domain containing proteins involved in cell signaling and vesicular transport. Among others, the interaction between tyrosine‐phosphorylated CTFs and ShcA‐Grb2 adaptors is highly enhanced in AD brain. Here we have identified in SH‐SY5Y neuroblastoma cells an interaction between APP holoprotein and the adaptor Grb2. Upon activation of apoptotic cell death this interaction is rapidly degraded, APP is partially cleaved and the complex APP/Grb2 is replaced by a new complex between CTFs and ShcA that still involves Grb2. The formation of these complexes is regulated by beta‐site APP‐cleaving enzyme 1 and influences the phosphorylation of mitogen‐activated protein kinase p44/42 extracellular signal‐regulated kinase as well as the level of apoptotic death of the cells. These data suggest a dual role in cell signaling for APP and its CTFs in neuroblastoma cells, in a manner similar to that previously reported for other tyrosine kinase receptor, through a tightly regulated coupling with alternative intracellular adaptors to control the signaling of the cell.

Amyloid precursor protein modulates ERK-1 and -2 signaling.

Abstract:  The amyloid precursor protein (APP) is a transmembrane protein with a short cytoplasmic tail whose physiological function is unclear, although it is well documented that the proteolytic processing of APP could influence the development of Alzheimers disease (AD) through the formation of membrane‐bound C‐terminal fragments (CTFs) and of β‐amyloid peptides (Aβ). We have recently shown that tyrosine‐phosphorylated APP and CTFs may interact with Grb2 and ShcA adaptor proteins and that this coupling occurs at a higher extent in AD subjects only. To study the interaction between APP or CTFs and ShcA/Grb2 and to investigate their molecular target we have used as experimental model two different cell lines: H4 human neuroglioma cells and APP/APLP null mouse embryonic fibroblast cells (MEFs). Here we show that in H4 cells APP interacts with Grb2; conversely in APP/APLP‐null MEF cells this interaction is possible only after the reintroduction of human APP by transfection. We have also shown that in MEF cells the transfection of a plasmid encoding for human APP wild‐type enhances the phosphorylation of ERK‐1 and ‐2 as revealed by Western blotting and immunofluorescence experiments. Finally, also in H4 cells the overexpression of APP upregulates the levels of phospho‐ERK‐1 and ‐2. In summary our data suggest that APP may influence phospho‐ERK‐1 and ‐2 signaling through its binding with Grb2 and ShcA adaptors. The meaning of this event is not clear, but APP interaction with these adaptors could be relevant to regulate mitogenic pathway.

BACE1 overexpression regulates amyloid precursor protein cleavage and interaction with the ShcA adapter.

Abstract: The amyloid precursor protein (APP) is a cell surface protein with a large extracellular N‐terminal domain, a single transmembrane segment, and a short cytoplasmic tail. Its location and structural features are characteristic of a receptor for signal transduction. Yet, the physiological function of APP is unclear, although it is well documented that APPs proteolytic processing, through the formation of membrane‐bound C‐terminal fragments (CTFs) and of β‐amyloid peptides, likely influences the development of Alzheimers disease (AD). There is evidence that BACE1 is the enzyme responsible for β‐site cleavage of the APP and for the generation of CTFs. BACE1 expression is upregulated in AD brain, and we have recently shown in human brain and in vitro that BACE product CTFs, when phosphorylated in tyrosine residues, interact with the adaptor proteins ShcA and Grb2, which usually are involved in signal transduction pathways. We investigated the interaction between ShcA, APP, and CTFs in the H4 human cell line that overexpresses BACE1 to clarify the significance of such interactions in vitro and for AD generation. Our result show that the APP, CTF, and ShcA interaction is induced only upon overexpression of BACE1 either transiently or in stable cell lines. In particular, although BACE1 drives the formation of C99 and C89 CTFs, only C99 interacts with the ShcA adaptor protein. Therefore, our data suggest that BACE1 activity influences APP processing and its intracellular signaling through the ShcA adaptor protein.