Valentina Venezia

Signal Transduction through Tyrosine-phosphorylated C-terminal Fragments of Amyloid Precursor Protein via an Enhanced Interaction with Shc/Grb2 Adaptor Proteins in Reactive Astrocytes of Alzheimer's Disease Brain

The proteolytic processing of amyloid precursor protein (APP) through the formation of membrane-bound C-terminal fragments (CTFs) and of soluble β-amyloid peptides likely influences the development of Alzheimers disease (AD). We show that in human brain a subset of CTFs are tyrosine-phosphorylated and form stable complexes with the adaptor protein ShcA. Grb2 is also part of these complexes, which are present in higher amounts in AD than in control brains. ShcA immunoreactivity is also greatly enhanced in patients with AD and occurs at reactive astrocytes surrounding cerebral vessels and amyloid plaques. A higher amount of phospho-ERK1,2, likely as result of the ShcA activation, is present in AD brains. In vitroexperiments show that the ShcA-CTFs interaction is strictly confined to glial cells when treated with thrombin, which is a well known ShcA and ERK1,2 activator and a regulator of APP cleavage. In untreated cells ShcA does not interact with either APP or CTFs, although they are normally generated. Altogether these data suggest that CTFs are implicated in cell signaling via Shc transduction machinery, likely influencing MAPK activity and glial reaction in AD patients.

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.

Identification of Amino-Terminally and Phosphotyrosine-Modified Carboxy-Terminal Fragments of the Amyloid Precursor Protein in Alzheimer's Disease and Down's Syndrome Brain

The carboxy-terminal fragments (CTFs) of the amyloid precursor protein (APP) are considered β-amyloid (Aβ) precursors as well as molecular species possibly amyloidogenic and neurotoxic by themselves in vitro or in animal models. The CTFs role in the pathogenesis of Alzheimers disease (AD) is however relatively unexplored in human brain. In this study, we analyzed brain extracted CTFs in subjects with AD, non-AD control, and Downs syndrome (DS) cases. Our data indicate that: (i) In fetal DS subjects CTFs levels are increased in comparison to age-matched control, suggesting that the enhanced CTFs formation is important for the early occurrence of plaques deposition in DS. No significant difference in CTFs level is present between AD and age-matched control cases. (ii) CTFs modified at their N-terminus are the direct precursors of similarly N-terminally modified Aβ peptides, which constitute the most abundant species in AD and DS plaques. This observation suggests that N-truncated Aβ peptides are formed directly at β-secretase level and not through a progressive proteolysis of full-length Aβ1-40/42. (iii) Among the differently cleaved CTFs, only the 22- and 12.5-kDa CTF polypeptides are tyrosine phosphorylated in both AD and control brain while the full-length APP and the CTFs migrating below the 12.5-kDa marker are not phosphorylated, suggesting that APP and CTFs may be involved in different pathways depending on their length and sequences. This study provides evidence that CTFs constitute in human brain a molecular species directly involved in AD pathogenesis and in the development of the AD-like pathology in DS subjects.

ERK1/2 and p38 MAP Kinases Control Prion Protein Fragment 90-231-Induced Astrocyte Proliferation and Microglia Activation

Astrogliosis and microglial activation are a common feature during prion diseases, causing the release of chemoattractant and proinflammatory factors as well as reactive free radicals, involved in neuronal degeneration. The recombinant protease‐resistant domain of the prion protein (PrP90–231) displays in vitro neurotoxic properties when refolded in a β‐sheet‐rich conformer. Here, we report that PrP90–231 induces the secretion of several cytokines, chemokines, and nitric oxide (NO) release, in both type I astrocytes and microglial cells. PrP90–231 elicited in both cell types the activation of ERK1/2 MAP kinase that displays, in astrocytes, a rapid kinetics and a proliferative response. Conversely, in microglia, PrP90–231‐dependent MAP kinase activation was delayed and long lasting, inducing functional activation and growth arrest. In microglial cells, NO release, dependent on the expression of the inducible NO synthase (iNOS), and the secretion of the chemokine CCL5 were Ca2+ dependent and under the control of the MAP kinases ERK1/2 and p38: ERK1/2 inhibition, using PD98059, reduced iNOS expression, while p38 blockade by PD169316 inhibited CCL5 release. In summary, we demonstrate that glial cells are activated by extracellular misfolded PrP90–231 resulting in a proliferative/secretive response of astrocytes and functional activation of microglia, both dependent on MAP kinase activation. In particular, in microglia, PrP90–231 activated a complex signalling cascade involved in the regulation of NO and chemokine release. These data argue in favor of a causal role for misfolded prion protein in sustaining glial activation and, possibly, glia‐mediated neuronal death.

Signal transduction through tyrosine-phosphorylated carboxy-terminal fragments of APP via an enhanced interaction with Shc/Grb2 adaptor proteins in reactive astrocytes of Alzheimer's disease brain.

Abstract: The processing of the amyloid precursor protein (APP) through the formation of C‐terminal fragments (CTFs) and the production of β‐amyloid, are events likely to influence the development and the progression of Alzheimers disease (AD). APP is a transmembrane protein similar to a cell‐surface receptor with the intraluminal NPTY motif in the cytosolic C terminus. Although APP holoprotein can be bound to intracellular proteins like Fe65, X11, and mDab, the ultimate function and the mechanisms through which this putative receptor transfers its message are unclear. Here it is shown that in human brain, a subset of tyrosine‐phosphorylated CTFs represent docking sites for the adaptor protein ShcA. ShcA immunoreactivity is greatly enhanced in Alzheimers patients; it is mainly localized to glial cells and occurs at reactive astrocytes surrounding cerebral vessels and amyloid plaques. Grb2 also is involved in complexes with ShcA and tyrosine‐phosphorylated CTFs, and in AD brain the interaction between Grb2‐ShcA and CTFs is enhanced. Also, a higher amount of phospho‐ERK1,2 is present in AD brain in comparison with control cases, likely as a result of the ShcA activation. In vitro experiments show that the ShcA‐CTFs interaction is strictly confined to glial cells when treated with thrombin, which is a well‐known ShcA and ERK1,2 activator, mitogen, and regulator of APP cleavage. In untreated cells ShcA does not interact with either APP or CTFs, although they are normally produced. Altogether these data suggest that CTFs are implicated in cell signaling via Shc transduction machinery, likely influencing MAPK activity and glial reaction in AD patients.

Characterization of the Proapoptotic Intracellular Mechanisms Induced by a Toxic Conformer of the Recombinant Human Prion Protein Fragment 90–231

Abstract:  Prion diseases comprise a group of fatal neurodegenerative disorders that affect both animals and humans. The transition of the prion protein (PrP) from a mainly α‐structured isoform (PrPC) to a prevalent β‐sheet‐containing protein (PrPSc) is believed to represent a major pathogenetic mechanism in prion diseases. To investigate the linkage between PrP neurotoxicity and its conformation, we used a recombinant prion protein fragment corresponding to the amino acidic sequence 90–231 of human prion protein (hPrP90–231). Using thermal denaturation, we set up an experimental model to induce the process of conversion from PrPC to PrPSc. We report that partial thermal denaturation converts hPrP90–231 into a β‐sheet‐rich isoform, displaying a temperature‐ and time‐dependent conversion into oligomeric structures that share some physico‐chemical characteristics with brain PrPSc. SH‐SY5Y cells were chosen to characterize the potential neurotoxic effect of hPrP90–231 in its different structural conformations. We demonstrated that hPrP90–231 in β‐conformation, but not when α‐structured, powerfully affected the survival of these cells. hPrP90–231 β‐structured caused DNA fragmentation and a significant increase in caspase‐3 proteolytic activity (maximal effects + 170%), suggesting the occurrence of apoptotic cell death. Finally, we investigated the involvement of MAP kinases in the regulation of β‐hPrP90–231‐dependent apoptosis. We observed that the p38 MAP kinase blocker SB203580 prevented the apoptotic cell death evoked by hPrP90–231, and Western blot analysis revealed that the exposure of the cells to the peptide induced p38 phosphorylation. In conclusion, we demonstrate that the hPrP90–231 elicits proapoptotic activity when in β‐sheet‐rich conformation and that this effect is mediated by p38 and caspase‐3 activation.

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 and Presenilin Involvement in Cell Signaling

To date the most relevant role for the amyloid precursor protein (APP) and for the presenilins (PSs) on Alzheimer’s disease (AD) genesis is linked to the ‘amyloid hypothesis’, which considers an aberrant formation of amyloid-β peptides the cause of neurodegeneration. In this view, APP is merely a substrate, cleaved by the γ-secretase complex to form toxic amyloid peptides, PSs are key players in γ-secretase complex, and corollary or secondary events are Tau-linked pathology and gliosis. A second theory, complementary to the amyloid hypothesis, proposes that APP and PSs may modulate a yet unclear cell signal, the disruption of which may induce cell-cycle abnormalities, neuronal death, eventually amyloid formation and finally dementia. This hypothesis is supported by the presence of a complex network of proteins, with a clear relevance for signal transduction mechanisms, which interact with APP or PSs. In this scenario, the C-terminal domain of APP has a pivotal role due to the presence of the 682YENPTY687 motif that represents the docking site for multiple interacting proteins involved in cell signaling. In this review we discuss the significance of novel findings related to cell signaling events modulated by APP and PSs for AD development.

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.

Apoptotic Cell Death and Amyloid Precursor Protein Signaling in Neuroblastoma SH‐SY5Y Cells

Abstract: We have recently shown that the amyloid precursor protein (APP) and a subset of its C‐terminal fragments (CTFs) are tyrosine phosphorylated in human brain and in cultured cells. Tyrosine phosphorylation generates a substrate that is sequentially bound by the adaptor proteins ShcA and Grb2, and this interaction is significantly enhanced in Alzheimers disease brains. Here we have studied the APP/CTFs phosphorylation and ShcA activation in a human neuroblastoma cell line, SH‐SY5Y, under basal and apoptotic conditions. To commit these cells to apoptosis, we used staurosporin, a well‐known apoptotic inducer and protein kinase C blocker. Our data suggest the following: (1) in normally proliferating SH‐SY5Y cells, full‐length APP is complexed with Grb2[Q3], likely through its SH2 domain; (2) upon induction of apoptosis, APP is degraded and ShcA‐Grb2 coimmunoprecipitates with CTFs recognized by anti‐APP antibodies; and (3) caspase inhibitors partially block the degradation of APP and the coprecipitation of CTFs with ShcA‐Grb2 adaptors. In summary, our data suggest that in SH‐SY5Y cells, tyrosine‐phosphorylated APP is involved in a complex with ShcA‐Grb2 adaptors that is disrupted during apoptosis. The abnormal degradation of APP and consequent increased levels of CTFs (as has been observed in Alzheimers disease and Downs syndrome) generate a complex between tyrosine‐phosphorylated CTFs and intracellular adaptors. The signaling through APP and its CTFs may have significant relevance for apoptotic cell death in Alzheimers disease.